Homogenous suspension of immunopotentiating compounds and uses thereof

ABSTRACT

The present invention generally relates to homogeneous suspensions of small molecule immune potentiators (SMIPs) that are capable of stimulating or modulating an immune response in a subject in need thereof. The homogeneous suspensions may be used in combinations with various antigens or adjuvants for vaccine therapies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/US2010/060621, filed Dec. 15, 2010 and published in English,which claims the benefit of U.S. Provisional Application No. 61/286,754,filed Dec. 15, 2009, which applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Oct. 24, 2012, is namedSeq_List.TXT, and is 29,006 bytes in size.

BACKGROUND OF THE INVENTION

Recently developed attenuated pathogen or subunit protein vaccines,while offering significant advantages over the traditional wholepathogen vaccines in terms of safety and cost of production, generallyhave limited immunogenicity as compared to whole pathogens. As a result,these vaccines typically require adjuvants with significantimmunostimulatory capability to reach their full potential in preventingdiseases.

Efforts have been made to identify new immune modulators for use asadjuvants for vaccines and immunotherapies. In particular, an adjuvantformulation that elicits potent cell-mediated and humoral immuneresponses to a wide range of antigens in humans and domestic animals,but lacking the side effects of conventional adjuvants and other immunemodulators, would be highly desirable. This need could be met by smallmolecule immune potentiators (“SMIPs”) because the small moleculeplatform provides diverse compounds for the selective manipulation ofthe immune response, necessary for increasing the therapeutic indeximmune modulators.

Toll-like receptors (TLRs) are a group of pattern recognition receptorswhich bind to pathogen-associated molecular patterns (PAMPS) frombacteria, fungi, protozoa and viruses, and act as a first line ofdefense against invading pathogens. TLRs are essential to induceexpression of genes involved in inflammatory responses, and TLRs and theinnate immune system are a critical step in the development ofantigen-specific acquired immunity.

Adaptive (humoral or cell-mediated) immunity is associated with the TLRsignal mechanism of innate immunity. Innate immunity is a protectiveimmune cell response that functions rapidly to fight environmentalinsults including, but not limited to, bacterial or viral agents.Adaptive immunity is a slower response, which involves differentiationand activation of naive T lymphocytes into T helper 1 (Th1) or T helper2 (Th2) cell types. Th1 cells mainly promote cellular immunity, whereasTh2 cells mainly promote humoral immunity.

All TLRs appear to function as either a homodimer or heterodimer in therecognition of a specific, or set of specific, molecular determinantspresent on pathogenic organisms including bacterial cell-surfacelipopolysaccharides, lipoproteins, bacterial flagellin, DNA from bothbacteria and viruses and viral RNA. The cellular response to TLRactivation involves activation of one or more transcription factors,leading to the production and secretion of cytokines and co-stimulatorymolecules such as interferons, TNF-, interleukins, MIP-1 and MCP-1 whichcontribute to the killing and clearance of the pathogenic invasion.

Thirteen TLRs (named TLR1 to TLR13) have been identified in humans andmice together, and equivalent forms of many of these have been found inother mammalian species. In particular, the roles of TLR7 and TLR8 areto detect the presence of “foreign” single-stranded RNA within a cell,as a means to respond to viral invasion. Both TLR7 and TLR8 arestructurally highly conserved proteins that recognize guanosine- oruridine-rich, single-stranded RNA (ssRNA) from viruses such as humanimmunodeficiency virus, vesicular stomatitis virus and influenza virus.

WO 2009/111337 discloses a series of compounds that bind to Toll-LikeReceptors (TLR), including TLR7 and TLR8. The compounds are found to beuseful as immunopotentiators, but contain a hydrophobic core and havelow solubility.

It is therefore an object of this invention to provide homogeneoussuspensions of SMIPs that are capable of stimulating or modulating animmune response in a subject in need thereof.

SUMMARY OF THE INVENTION

The present invention generally relates to homogeneous suspensions ofsmall molecule immune potentiators (SMIPs; i.e. compounds of Formula Ior Formula II) that are capable of stimulating or modulating an immuneresponse in a subject in need thereof. The homogeneous suspensions maybe used in combinations with various antigens or adjuvants forimmunotherapy.

One exemplary approach is to use high pressure homogenization togetherwith a surfactant and a viscosity-enhancing agent to create a stablehomogeneous suspension of SMIP that contains particles in μm size range.The resulting suspensions are particularly suitable for co-delivery ofSMIP with alum and oil-in-water emulsion based vaccines.

In one aspect, the invention provides a homogeneous suspensioncomprising (a) a Benzonaphthyridine compound of Formula I or Formula II,pharmaceutically acceptable salt, pharmaceutically acceptable solvate,N-oxide derivative, protected derivative, individual isomer or mixtureof isomers thereof; (b) a surfactant, and (c) a suspension agent,wherein the suspension is stable for at least about four weeks at 4° C.In certain embodiments, the homogeneous suspension comprises about 0.5mg/mL to about 50 mg/mL Benzonaphthyridine compound.

In certain embodiments, the Benzonaphthyridine compound is:2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol;2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate;2-(4-(dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine; or2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol.

In a preferred embodiment, the homogeneous suspension comprises (a)2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine, or apharmaceutically acceptable salt or pharmaceutically acceptable solvatethereof, (b) a surfactant, and (c) a suspension agent, wherein thesuspension is stable for at least about four weeks at 4° C.

In another preferred embodiment, the homogeneous suspension comprises(a)2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine,or a pharmaceutically acceptable salt or pharmaceutically acceptablesolvate thereof, (b) a surfactant, and (c) a suspension agent, whereinthe suspension is stable for at least about four weeks at 4° C.

In another preferred embodiment, the homogeneous suspension comprises(a)2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethano1, or a pharmaceutically acceptable salt or pharmaceutically acceptablesolvate thereof, (b) a surfactant, and (c) a suspension agent, whereinthe suspension is stable for at least about four weeks at 4° C.

Exemplary surfactants include, e.g., Tween-80. In certain embodiments,the homogeneous suspension comprises about 0.1% to about 10% surfactant.Exemplary suspension agent includes, e.g., viscosity-enhancing agentssuch carboxymethyl cellulose. In certain embodiments, the homogeneoussuspension comprises about 0.1% to about 10% suspension agent.

In certain embodiments, at least about 50% of the suspension particlesof the homogeneous suspension have a diameter of about 10 μm or less. Incertain embodiments, at least about 50% of the suspension particles ofthe homogeneous suspension have a diameter of about 2 μm or less.

The homogeneous suspension of the invention can be used to potentiate animmune response and can be co-delivered with an antigen, immunogeniccomposition or vaccine to enhance the effectiveness of the inducedimmune response. An adjuvant (e.g., Alum or oil-in-water adjuvants) mayalso be used in combination with the homogeneous suspension of theinvention.

Also provided herein are immunogenic compositions comprising (1) anantigen and (2) the homogeneous suspension of the invention.

In certain embodiments, the immunogenic composition further comprises anadjuvant. Exemplary adjuvants include, e.g., an aluminum-containingadjuvant (e.g., aluminum hydroxide, aluminum oxyhydroxide, or aluminumhydroxyphosphate), an oil-in-water emulsion (e.g., MF59), a liposome(e.g., an outer membrane vesicle), an oligonucleotide (e.g., anoligonucleotide comprising an unmethylated CpG motif).

In certain embodiments, the immunogenic composition comprises an antigenthat is a bacterial antigen (e.g, an antigen from Neisseriameningitides), a viral antigen (e.g., an antigen from respiratorysyncytial virus (RSV) or Ebola virus). In certain embodiment, theimmunogenic composition comprises (1) a viral antigen (e.g., an antigenfrom Ebola virus) and (2)2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amineor2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol.

Also provided herein are methods of using the homogeneous suspension ofthe invention to potentiate an immune response in a subject in needthereof, and methods of producing the homogeneous suspension of theinvention.

The invention also relates to a method for producing a homogeneoussuspension that comprises a compound of Formula I or Formula II, asurfactant and a suspension agent. The method comprises mixing thecompound of Formula I or Formula II, a surfactant and a suspensionagent, and homogenizing the mixture under high pressure, such as15,000-20,000 psi, to produce a homogeneous suspension that preferablyhas a D50 of about 10 μm or less, more preferably, about 2 μm or less,and, optionally, a D90 of about 10 μm or less.

Also provided herein are methods of using the immunogenic composition ofthe invention to generating an immune response in a subject in needthereof.

The invention also relates to a homogeneous suspension as describedherein for use in therapy, and to the use of a homogeneous suspensionfor the manufacture of a medicament for potentiating or inducing animmune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the electrophoretic profiles (by SDS-PAGE) of three MenBantigens, alone or after adding different doses of Example 47 suspensionto the Alum/3MenB formulations. Key: SN=unadsorbed antigen(representative of 20% of 10 μg dose); T=unadsorbed antigen(representative of 200% of 20 μg dose); P=adsorbed antigen recovery(representative of 10% of 10 μg dose). FIG. 1B is a table showing thepercentage of antigen adsorption onto alum.

FIG. 2 shows that homogeneous suspension of Example 47 had no impact onthe adsorption of the extra-intestinal pathogenic E. coli (ExPEC)antigen PK1-3526. Key: 1. Sea Blue Plus MW Marker; 2. pK1-3526 control 2ug (100%); 3. pK1-3526 control 1 ug (50%); 4. pK1-3526 control 0.4 ug(20%); 5. Alum/pK1-3526 SN (20%); 6. Alum/pK1-3526+Example 47 SN (20%);7. Alum/pK1-3526 SN TCA (240%); 8. Alum/pK1-3526+Example 47 SN TCA(240%); 9.-; 10. pK1-3526 TCA control 2.4 ug (240%); 11. Alum/pK1-3526Pellet Desorb (50%); 12. Alum/pK1-3526+Example 47 Pellet Desorb (50%). %values indicate proportions of single dose formulation.

FIG. 3A shows the serum levels of Example 47 (non-homogencous/DMSOsuspension versus homogeneous suspension) in mice after intramuscularinjection at 4 mpk (miligram per kilogram) in the vehicles indicatedwith 3MenB/Alum. FIG. 3B shows the injection site muscle levels ofExample 47 (non-homogeneous/DMSO suspension versus homogeneoussuspension) in mice 24 hours after intramuscular injection at 4 mpk inthe vehicles indicated with 3MenB/Alum. FIG. 3C shows inguinal lymphnode level of Example 47 (homogeneous suspension) in mice 24 hours afterintramuscular injection at 4 mpk in the vehicles indicated with3MenB/Alum.

FIG. 4 shows that cytokine release profiles of non-homogeneous/DMSOsuspension plus 3MenB/Alum and homogeneous suspension of Example 47 plus3MenB/Alum were similar. Cytokine releases were minimal as compared toadjuvant R848 after exposure of homogeneous suspensions or DMSOformulated Example 47.

FIG. 5A shows the serum bactericidal antibody (SBA) titers forAlum/3MenB formulated with specified doses of Example 47 homogeneoussuspension ranging from 2 to 100 microgram per mouse. FIG. 5B shows theSBA titers for MF59/3MenB formulated with specified doses of Example 47homogeneous suspension ranging from 2 to 100 microgram per mouse.

FIG. 6 shows the ability of the Example 47 suspension (when formulatedwith Alum/3MenB vaccine) to elicit a SBA titer for pooled sera against apanel of 16 stains of N. meningitides (all of which had typically lowtiters with Alum/3MenB formulations). The graph depicts the percentageof coverage against these 16 strains. Cut-off refers to the lowesttiters considered to show bactericidal activity.

FIGS. 7A-7C show the result of evaluation of IgG1/IgG2a ratios in theMenB mouse model in vivo. FIG. 7A shows the IgG1, IgG2a, IgG2b and IgG3titres for GNA2132-GNA1030. FIG. 7B shows the IgG1, IgG2a, IgG2b andIgG3 titres for GNA2091-GNA1870. FIG. 7C shows the IgG1, IgG2a, IgG2band IgG3 titres for NadA.

FIG. 8 shows the results of SBA assays using the Example 47non-homogeneous suspension (prepared in DMSO). Mice were immunized twicevia i.m. with MF59/3MenB+the indicated amount of Example 47. The graphis based on 3 independent SBA studies and depicts average titer±standarddeviation at two weeks post-2nd injection. SBA assays were performedwith standard CFU method with NZ98 MenB strain.

FIG. 9A is a graph summarizing the results of one study thatdemonstrated the ability of the Example 47 suspension to elicit animmune response against RSV F-antigen. The readouts represent totalanti-F antibody (kappa) and neutralizing antibody (PRNT60).

FIG. 9B shows that in another study, the Example 47 suspension enhancedF-specific antibody, class switching to IgG2a, and neutralizingantibody.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

In one aspect, this invention relates to homogeneous suspension of smallmolecule immune potentiators (SMIPs). The homogeneous suspensions can beused to potentiate an immune response.

As described herein, the inventors have evaluated the efficacy ofhomogeneous suspensions of an exemplary SMIP,2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine. Thesestudies demonstrated that homogeneous suspensions of SMIP are moreeffective than non-homogeneous suspensions (in DMSO) in potentiatingimmune response.

One exemplary approach to produce the homogeneous suspensions is to usehigh pressure homogenization together with a surfactant and aviscosity-enhancing agent to create a stable homogeneous suspension ofSMIP that contains particles in the 100 nm to 10 μm size range. Theresulting suspensions are particularly suitable for co-delivery of SMIPwith alum and oil-in-water emulsion based vaccines. A homogeneoussuspension has a defined size distribution, allowing better uniformityof doses. Furthermore, the resulting particle size increasesbioavailability of the SMIP to immune cells post administration. Thesuspensions also have the potential to be stable over time as measuredby size distribution. Finally, a homogeneous suspension of SMIP provideimmuno-stimulatory effect at lower doses (e.g., 10 μg/dose or less) ascompared to a non-homogeneous suspension at the same dosage.

In a preferred embodiment, the homogeneous suspension comprises (a)2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine, orpharmaceutically acceptable salt or pharmaceutically acceptable solvatethereof, (b) a surfactant, and (c) a suspension agent, wherein thesuspension is stable for at least about four weeks at 4° C.

In another preferred embodiment, the homogeneous suspension comprises(a)2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine,or a pharmaceutically acceptable salt or pharmaceutically acceptablesolvate thereof, (b) a surfactant, and (c) a suspension agent, whereinthe suspension is stable for at least about four weeks at 4° C.

In another preferred embodiment, the homogeneous suspension comprises(a)2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol,or a pharmaceutically acceptable salt or pharmaceutically acceptablesolvate thereof, (b) a surfactant, and (c) a suspension agent, whereinthe suspension is stable for at least about four weeks at 4° C.

The homogeneous suspension of the invention can be used to potentiate animmune response and can be co-delivered with an antigen, immunogeniccomposition or vaccine to enhance the effectiveness of the inducedimmune response.

Also provided herein are methods of using the homogeneous suspension ofthe invention to potentiate an immune response in a subject in needthereof, and methods of producing the homogeneous suspension of theinvention.

2. Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise.

The term “about”, as used here, refers to +/−10% of a value.

The term “alkenyl,” as used herein, refers to a partially unsaturatedbranched or straight chain hydrocarbon having at least one carbon-carbondouble bond. Atoms oriented about the double bond are in either the cis(Z) or trans (E) conformation. An alkenyl group can be optionallysubstituted. As used herein, the terms “C₂-C₃alkenyl”, “C₂-C₄alkenyl”,“C₂-C₂alkenyl”, “C₂-C₆alkenyl”, “C₂-C₇alkenyl”, and “C₂-C₈alkenyl” referto an alkenyl group containing at least 2, and at most 3, 4, 5, 6, 7 or8 carbon atoms, respectively. If not otherwise specified, an alkenylgroup generally is a C₂-C₆ alkenyl. Non-limiting examples of alkenylgroups, as used herein, include ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl and the like.

The term “alkenylene,” as used herein, refers to a partially unsaturatedbranched or straight chain divalent hydrocarbon radical derived from analkenyl group. An alkenylene group can be optionally substituted. Asused herein, the terms “C₂-C₃alkenylene”, “C₂-C₄alkenylene”,“C₂-C₅alkenylene”, “C₂-C₆alkenylene”, “C₂-C₇alkenylene”, and“C₂-C₈alkenylene” refer to an alkenylene group containing at least 2,and at most 3, 4, 5, 6, 7 or 8 carbon atoms respectively. If nototherwise specified, an alkenylene group generally is a C₁-C₆alkenylene. Non-limiting examples of alkenylene groups as used hereininclude, ethenylene, propenylene, butenylene, pentenylene, hexenylene,heptenylene, octenylene, nonenylene, decenylene and the like.

The term “alkyl,” as used herein, refers to a saturated branched orstraight chain hydrocarbon. An alkyl group can be optionallysubstituted. As used herein, the terms “C₁-C₃alkyl”, “C₁-C₄alkyl”,“C₁-C₅alkyl”, “C₁-C₆alkyl”, “C₁-C₇alkyl” and “C₁-C₈alkyl” refer to analkyl group containing at least 1, and at most 3, 4, 5, 6, 7 or 8 carbonatoms, respectively. If not otherwise specified, an alkyl groupgenerally is a C₁-C₆ alkyl. Non-limiting examples of alkyl groups asused herein include methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl,nonyl, decyl and the like.

The term “alkylene,” as used herein, refers to a saturated branched orstraight chain divalent hydrocarbon radical derived from an alkyl group.An alkylene group can be optionally substituted. As used herein, theterms “C₁-C₃alkylene”, “C₁-C₄alkylene”, “C₁-C₅alkylene”,“C₁-C₆alkylene”, “C₁-C₇alkylene” and “C₁-C₈alkylene” refer to analkylene group containing at least 1, and at most 3, 4, 5, 6, 7 or 8carbon atoms respectively. If not otherwise specified, an alkylene groupgenerally is a C₁-C₆ alkylene. Non-limiting examples of alkylene groupsas used herein include, methylene, ethylene, n-propylene, isopropylene,n-butylene, isobutylene, sec-butylene, t-butylene, n-pentylene,isopentylene, hexylene and the like.

The term “alkynyl,” as used herein, refers to a partially unsaturatedbranched or straight chain hydrocarbon having at least one carbon-carbontriple bond. An alkynyl group can be optionally substituted. As usedherein, the terms “C₂-C₃alkynyl”, “C₂-C₄alkynyl”, “C₂-C₅alkynyl”,“C₂-C₆alkynyl”, “C₂-C₇alkynyl”, and “C₂-C₈alkynyl” refer to an alkynylgroup containing at least 2, and at most 3, 4, 5, 6, 7 or 8 carbonatoms, respectively. If not otherwise specified, an alkynyl groupgenerally is a C₂-C₆ alkynyl. Non-limiting examples of alkynyl groups,as used herein, include ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl and the like.

The term “alkynylene,” as used herein, refers to a partially unsaturatedbranched or straight chain divalent hydrocarbon radical derived from analkynyl group. An alkynylene group can be optionally substituted. Asused herein, the terms “C₂-C₃alkynylene”, “C₂-C₄alkynylene”,“C₂-C₅alkynylene”, “C₂-C₆alkynylene”, “C₂-C₇alkynylene”, and“C₂-C₈alkynylene” refer to an alkynylene group containing at least 2,and at most 3, 4, 5, 6, 7 or 8 carbon atoms respectively. If nototherwise specified, an alkynylene group generally is a C₂-C₆alkynylene. Non-limiting examples of alkynylene groups as used hereininclude, ethynylene, propynylene, butynylene, pentynylene, hexynylene,heptynylene, octynylene, nonynylene, decynylene and the like.

The term “alkoxy,” as used herein, refers to the group —OR_(a), whereR_(a) is an alkyl group as defined herein. An alkoxy group can beoptionally substituted. As used herein, the terms “C₁-C₃alkoxy”,“C₁-C₄alkoxy”, “C₁-C₅alkoxy”, “C₁-C₆alkoxy”, “C₁-C₇alkoxy” and“C₁-C₈alkoxy” refer to an alkoxy group wherein the alkyl moiety containsat least 1, and at most 3, 4, 5, 6, 7 or 8, carbon atoms. Non-limitingexamples of alkoxy groups, as used herein, include methoxy, ethoxy,n-propoxy, isopropoxy, n-butyloxy, t-butyloxy, pentyloxy, hexyloxy,heptyloxy, octyloxy, nonyloxy, decyloxy and the like.

The term “aryl,” as used herein, refers to monocyclic, bicyclic, andtricyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. An aryl group can beoptionally substituted. Non-limiting examples of aryl groups, as usedherein, include phenyl, naphthyl, fluorenyl, indenyl, azulenyl,anthracenyl and the like.

The term “arylene,” as used means a divalent radical derived from anaryl group. An arylene group can be optionally substituted.

The term “cyano,” as used herein, refers to a —CN group.

The term “cycloalkyl,” as used herein, refers to a saturated orpartially unsaturated, monocyclic, fused bicyclic, fused tricyclic orbridged polycyclic ring assembly. As used herein, the terms “C₃-C₅cycloalkyl”, “C₃-C₆ cycloalkyl”, “C₃-C₇ cycloalkyl”, “C₃-C₈ cycloalkyl,“C₃-C₉ cycloalkyl and “C₃-C₁₀ cycloalkyl refer to a cycloalkyl groupwherein the saturated or partially unsaturated, monocyclic, fusedbicyclic or bridged polycyclic ring assembly contain at least 3, and atmost 5, 6, 7, 8, 9 or 10, carbon atoms. A cycloalkyl group can beoptionally substituted. Non-limiting examples of cycloalkyl groups, asused herein, include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl,cyclohexenyl, decahydronaphthalenyl, 2,3,4,5,6,7-hexahydro-1H-indenyland the like.

The term “halogen,” as used herein, refers to fluorine (F), chlorine(Cl), bromine (Br), or iodine (I).

The term “halo,” as used herein, refers to the halogen radicals: fluoro(—F), chloro (—Cl), bromo (—Br), and iodo (—I).

The terms “haloalkyl” or “halo-substituted alkyl,” as used herein,refers to an alkyl group as defined herein, substituted with one or morehalogen groups, wherein the halogen groups are the same or different. Ahaloalkyl group can be optionally substituted. Non-limiting examples ofsuch branched or straight chained haloalkyl groups, as used herein,include methyl, ethyl, propyl, isopropyl, isobutyl and n-butylsubstituted with one or more halogen groups, wherein the halogen groupsare the same or different, including, but not limited to,trifluoromethyl, pentafluoroethyl, and the like.

The terms “haloalkenyl” or “halo-substituted alkenyl,” as used herein,refers to an alkenyl group as defined herein, substituted with one ormore halogen groups, wherein the halogen groups are the same ordifferent. A haloalkenyl group can be optionally substituted.Non-limiting examples of such branched or straight chained haloalkenylgroups, as used herein, include ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl and the like substitutedwith one or more halogen groups, wherein the halogen groups are the sameor different.

The terms “haloalkynyl” or “halo-substituted alkynyl,” as used herein,refers to an alkynyl group as defined above, substituted with one ormore halogen groups, wherein the halogen groups are the same ordifferent. A haloalkynyl group can be optionally substituted.Non-limiting examples of such branched or straight chained haloalkynylgroups, as used herein, include ethynyl, propynyl, butynyl, pentynyl,hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like substitutedwith one or more halogen groups, wherein the halogen groups are the sameor different.

The term “haloalkoxy,” as used herein, refers to an alkoxy group asdefined herein, substituted with one or more halogen groups, wherein thehalogen groups are the same or different. A haloalkoxy group can beoptionally substituted. Non-limiting examples of such branched orstraight chained haloalkynyl groups, as used herein, include methoxy,ethoxy, n-propoxy, isopropoxy, n-butyloxy, t-butyloxy, pentyloxy,hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy and the like,substituted with one or more halogen groups, wherein the halogen groupsare the same or different.

The term “heteroalkyl,” as used herein, refers to an alkyl group asdefined herein wherein one or more carbon atoms are independentlyreplaced by one or more of oxygen, sulfur, nitrogen, or combinationsthereof.

The term “heteroaryl,” as used herein, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms selected fromnitrogen, oxygen and sulfur, and wherein each ring in the systemcontains 3 to 7 ring members. A heteroaryl group may contain one or moresubstituents. A heteroaryl group can be optionally substituted.Non-limiting examples of heteroaryl groups, as used herein, includebenzofuranyl, benzofurazanyl, benzoxazolyl, benzopyranyl, benzthiazolyl,benzothienyl, benzazepinyl, benzimidazolyl, benzothiopyranyl,benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thienyl, cinnolinyl,furazanyl, furyl, furopyridinyl, imidazolyl, indolyl, indolizinyl,indolin-2-one, indazolyl, isoindolyl, isoquinolinyl, isoxazolyl,isothiazolyl, 1,8-naphthyridinyl, oxazolyl, oxaindolyl, oxadiazolyl,pyrazolyl, pyrrolyl, phthalazinyl, pteridinyl, purinyl, pyridyl,pyridazinyl, pyrazinyl, pyrimidinyl, quinoxalinyl, quinolinyl,quinazolinyl, 4H-quinolizinyl, thiazolyl, thiadiazolyl, thienyl,triazinyl, triazolyl and tetrazolyl.

The term “heterocycloalkyl,” as used herein, refers to a cycloalkyl, asdefined herein, wherein one or more of the ring carbons are replaced bya moiety selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—,wherein R is hydrogen, C₁-C₄alkyl or a nitrogen protecting group, withthe proviso that the ring of said group does not contain two adjacent Oor S atoms. A heterocycloalkyl group can be optionally substituted.Non-limiting examples of heterocycloalkyl groups, as used herein,include morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl,piperidinyl, piperidinylone, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl,2H-pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, 1,3-dioxolanyl, 2-imidazolinyl,imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl, 1,4-dioxanyl,1,4-dithianyl, thiomorpholinyl, azepanyl, hexahydro-1,4-diazepinyl,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,dihydropyranyl, tetrahydrothiopyranyl, thioxanyl, azetidinyl, oxetanyl,thietanyl, oxepanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, dithianyl, dithiolanyl,dihydropyranyl, dihydrothienyl, dihydrofuranyl, imidazolinyl,imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, and3-azabicyclo[4.1.0]heptanyl.

The term “heteroatom,” as used herein, refers to one or more of oxygen,sulfur, nitrogen, phosphorus, or silicon.

The term “hydroxyl,” as used herein, refers to the group —OH.

The term “hydroxyalkyl,” as used herein refers to an alkyl group asdefined herein substituted with one or more hydroxyl group. Non-limitingexamples of branched or straight chained “C₁-C₆ hydroxyalkyl groups asused herein include methyl, ethyl, propyl, isopropyl, isobutyl andn-butyl groups substituted with one or more hydroxyl groups.

The term “isocyanato,” as used herein, refers to a —N═C═O group.

The term “isothiocyanato,” as used herein, refers to a —N═C═S group

The term “mercaptyl,” as used herein, refers to an (alkyl)S— group.

The term “optionally substituted,” as used herein, means that thereferenced group may or may not be substituted with one or moreadditional group(s) individually and independently selected from alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl,hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl, thiocarbonyl,isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl,perfluoroalkyl, and amino, including mono- and di-substituted aminogroups, and the protected derivatives thereof. Non-limiting examples ofoptional substituents include, halo, —CN, ═O, ═N—OH, ═N—OR, ═N—R, —OR,—C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)NHR, —C(O)NR₂, —OC(O)NHR,—OC(O)NR₂, —SR—, —S(O)R, —S(O)₂R, —NHR, —N(R)₂, —NHC(O)R, —NRC(O)R,—NHC(O)OR, —NRC(O)OR, S(O)₂NHR, —S(O)₂N(R)₂, —NHS(O)₂NR₂, —NRS(O)₂NR₂,—NHS(O)₂R, —NRS(O)₂R, C₁-C₈alkyl, C₁-C₈alkoxy, aryl, heteroaryl,cycloalkyl, heterocycloalkyl, halo-substituted C₁-C₈alkyl, andhalo-substituted C₁-C₈alkoxy, where each R is independently selectedfrom H, halo, C₁-C₈alkyl, C₁-C₈alkoxy, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, halo-substituted C₁-C₈alkyl, and halo-substitutedC₁-C₈alkoxy. The placement and number of such substituent groups is donein accordance with the well-understood valence limitations of eachgroup, for example ═O is a suitable substituent for an alkyl group butnot for an aryl group.

The term “prodrug,” as used herein, refers to an agent that is convertedinto the parent drug in vivo. A non-limiting example of a prodrug of thecompounds described herein is a compound described herein administeredas an ester which is then metabolically hydrolyzed to a carboxylic acid,the active entity, once inside the cell. A further example of a prodrugis a short peptide bonded to an acid group where the peptide ismetabolized to reveal the active moiety.

The term “solvate,” as used herein, refers to a complex of variablestoichiometry formed by a solute (by way of example, a compound ofFormula (I), or a salt thereof, as described herein) and a solvent.Non-limiting examples of a solvent are water, acetone, methanol, ethanoland acetic acid.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered.

As used herein, “treatment” refers to any of (i) the prevention of acondition (e.g., a disease or disorder) in question (e.g. cancer or apathogenic infection, as in a traditional vaccine), (ii) the reductionor elimination of symptoms associated with the condition in question,and (iii) the substantial or complete elimination of the condition inquestion. Treatment may be effected prophylactically (prior to arrivalof the condition in question) or therapeutically (following arrival ofthe same).

The terms “effective amount” or “pharmaceutically effective amount” of ahomogeneous suspension of the present invention refers to an amountsufficient to potentiate an immune response, for example by at leastabout 10%, as described herein. The terms “effective amount” or“pharmaceutically effective amount” of an immunogenic composition of thepresent invention refer herein to a sufficient amount of the immunogeniccomposition for the treatment or diagnosis of a condition of interest.The exact amount required will vary from subject to subject, depending,for example, on the species, age, and general condition of the subject;the severity of the condition being treated; the particular antigen ofinterest; in the case of an immunological response, the capacity of thesubject's immune system to synthesize antibodies, for example, and thedegree of protection desired; and the mode of administration, amongother factors. An appropriate “effective” amount in any individual casemay be determined by one of ordinary skill in the art. Thus, a“therapeutically effective amount” will typically fall in a relativelybroad range that can be determined through routine trials.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,e.g., the material may be administered to an individual without causingany unduly undesirable biological effects or interacting in an undulydeleterious manner with any of the components of the composition inwhich it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 6.5 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used here, the term “injectable composition”, or variants thereof,refers to pharmaceutically acceptable compositions suitable forinjection into a vertebrate subject, which compositions are typicallysterile, pyrogen-free, and possess specific pH and isotonicity valuessuitable for injection.

As used herein, the term “particle” refers to a particle of about 10 nmor less to about 150 μm in diameter, for example, ranging from 10 nm to25 nm to 50 nm to 100 nm to 250 nm to 500 nm to 1 μm to 2.5 μm to 5 μmto 10 μm to 25 μm to 50 μm to 100 μm. The term “particle’ as used hereinthus includes “nanoparticle,” which is defined herein as a particlehaving a diameter less than 1000 nm, and “microparticle,” which isdefined herein as a particle having a diameter ranging from 1 μm to 1000μm. In some embodiments, the polymeric particles described herein can begenerally spherical.

The term “polypeptide” refers to a polymer of amino acid residues and isnot limited to a minimum length of the product. Thus, full lengthproteins, peptides, oligopeptides, dimers, multimers, and the like, areincluded within the definition.

A “polypeptide-containing species” is a molecule, at least a portion ofwhich is a polypeptide. Examples include polypeptides, proteinsincluding glycoproteins, metalloproteins and lipoproteins,polysaccharide antigens conjugated to carrier proteins, and so forth.Proteins for use herein include full-length proteins and fragmentsthereof. In certain embodiments, modifications to the native sequence,such as deletions, additions and substitutions (generally conservativein nature), are employed.

The term “fragment” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a fragment may be defined by a contiguous portion of the aminoacid sequence of that protein and may be at least 3-5 amino acids, atleast 6-10 amino acids, at least 11-15 amino acids, at least 16-24 aminoacids, at least 25-30 amino acids, and at least 30-45 amino acids. Inthe case of polynucleotide, a fragment is defined by a contiguousportion of the nucleic acid sequence of that polynucleotide and may beat least 9-15 nucleotides, at least 15-30 nucleotides, at least 31-45nucleotides, at least 46-74 nucleotides, at least 75-90 nucleotides, andat least 90-130 nucleotides. In some embodiments, fragments ofbiomolecules are immunogenic fragments.

A “polynucleotide” is a nucleic acid polymer. A polynucleotide caninclude as few as 5, 6, 7 or 8 nucleotides. Furthermore, a“polynucleotide” can include both double- and single-stranded sequencesand refers to, but is not limited to, cDNA from viral, procaryotic oreukaryotic mRNA, genomic RNA and DNA sequences from viral (e.g. RNA andDNA viruses and retroviruses), prokaryotic or eukaryotic organisms, andsynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA. The term further includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to a native sequence, for example, where thenucleic acid molecule encodes an antigenic protein. These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutations of hosts that produce antigens.

A “polynucleotide-containing species” is a molecule, at least a portionof which is a polynucleotide. Examples include RNA vector constructs,DNA vector constructs and so forth.

As used herein the term “saccharide” encompasses monosaccharides,oligosaccharides and polysaccharides. A “saccharide-containing species”is a molecule, at least a portion of which is a saccharide. Examplesinclude saccharide antigens, antigens comprising saccharides conjugatedto carrier peptides, and so forth.

As used herein the term “isolated” refers to a chemical species such asa polynucleotide, a polypeptide, and an antibody, etc. that is in anenvironment different from that in which the chemical species naturallyoccurs. A chemical species which is isolated is generally substantiallypurified. Methods of isolating cells are also well known to thoseskilled in the art.

A “purified” protein is a protein which is produced (e.g., recombinantlyor synthetically) or isolated from its natural host, such that theamount of protein present in a composition is substantially higher thanthat present in a crude preparation. In general, a purified protein willbe at least about 50% homogeneous, more preferably at least about 80%,about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, ormore, homogeneous.

As used herein, an “immunological response” or “immune response” is thedevelopment in a subject of a humoral and/or a cellular immune responseto the immunogenic species.

Immune responses include innate and adaptive immune responses. Innateimmune responses are fast-acting responses that provide a first line ofdefense for the immune system. In contrast, adaptive immunity usesselection and clonal expansion of immune cells having somaticallyrearranged receptor genes (e.g., T- and B-cell receptors) that recognizeantigens from a given pathogen or disorder (e.g., a tumor), therebyproviding specificity and immunological memory. Innate immune responses,among their many effects, lead to a rapid burst of inflammatorycytokines and activation of antigen-presenting cells (APCs) such asmacrophages and dendritic cells. To distinguish pathogens fromself-components, the innate immune system uses a variety of relativelyinvariable receptors that detect signatures from pathogens, known aspathogen-associated molecular patterns, or PAMPs. The addition ofmicrobial components to experimental vaccines is known to lead to thedevelopment of robust and durable adaptive immune responses. Themechanism behind this potentiation of the immune responses has beenreported to involve pattern-recognition receptors (PRRs), which aredifferentially expressed on a variety of immune cells, includingneutrophils, macrophages, dendritic cells, natural killer cells, B cellsand some nonimmune cells such as epithelial and endothelial cells.Engagement of PRRs leads to the activation of some of these cells andtheir secretion of cytokines and chemokines, as well as maturation andmigration of other cells. In tandem, this creates an inflammatoryenvironment that leads to the establishment of the adaptive immuneresponse. PRRs include nonphagocytic receptors, such as Toll-likereceptors (TLRs) and nucleotide-binding oligomerization domain (NOD)proteins, and receptors that induce phagocytosis, such as scavengerreceptors, mannose receptors and β-glucan receptors. Reported TLRs(along with examples of some reported ligands, which may be used asimmunogenic species in various embodiments of the invention) include thefollowing: TLR1 (bacterial lipoproteins from Mycobacteria, Neisseria),TLR2 (zymosan yeast particles, peptidoglycan, lipoproteins, glycolipids,lipopolysaccharide), TLR3 (viral double-stranded RNA, poly:IC), TLR4(bacterial lipopolysaccharides, plant product taxol), TLR5 (bacterialflagellins), TLR6 (yeast zymosan particles, lipotechoic acid,lipopeptides from mycoplasma), TLR7 (single-stranded RNA, imiquimod,resimiquimod, and other synthetic compounds such as loxoribine andbropirimine), TLR8 (single-stranded RNA, resimiquimod) and TLR9 (CpGoligonucleotides), among others. Dendritic cells are recognized as someof the most important cell types for initiating the priming of naiveCD4⁺ helper T (T_(H)) cells and for inducing CD8⁺ T cell differentiationinto killer cells. TLR signaling has been reported to play an importantrole in determining the quality of these helper T cell responses, forinstance, with the nature of the TLR signal determining the specifictype of T_(H) response that is observed (e.g., T_(H)1 versus T_(H)2response). A combination of antibody (humoral) and cellular immunity areproduced as part of a T_(H)1-type response, whereas a T_(H)2-typeresponse is predominantly an antibody response. Various TLR ligands suchas CpG DNA (TLR9) and imidazoquinolines (TLR7, TLR8) have beendocumented to stimulate cytokine production from immune cells in vitro.The imidazoquinolines are the first small, drug-like compounds shown tobe TLR agonists. For further information, see, e.g., A. Pashine, N. M.Valiante and J. B. Ulmer, Nature Medicine, 11, S63-S68 (2005), K. S.Rosenthal and D. H. Zimmerman, Clinical and Vaccine Immunology, 13(8),821-829 (2006), and the references cited therein.

For purposes of the present invention, a humoral immune response refersto an immune response mediated by antibody molecules, while a cellularimmune response is one mediated by T-lymphocytes and/or other whiteblood cells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (CTLs). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4⁺ and CD8⁺ T-cells.

A composition such as an immunogenic composition or a vaccine thatelicits a cellular immune response may thus serve to sensitize avertebrate subject by the presentation of antigen in association withMHC molecules at the cell surface. The cell-mediated immune response isdirected at, or near, cells presenting antigen at their surface. Inaddition, antigen-specific T-lymphocytes can be generated to allow forthe future protection of an immunized host. The ability of a particularantigen or composition to stimulate a cell-mediated immunologicalresponse may be determined by a number of assays known in the art, suchas by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, by assaying for T-lymphocytes specific for the antigen in asensitized subject, or by measurement of cytokine production by T cellsin response to restimulation with antigen. Such assays are well known inthe art. See, e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199;Doe et al. (1994) Eur. J. Immunol. 24:2369-2376. Thus, an immunologicalresponse as used herein may be one which stimulates the production ofCTLs and/or the production or activation of helper T-cells. The antigenof interest may also elicit an antibody-mediated immune response. Hence,an immunological response may include, for example, one or more of thefollowing effects among others: the production of antibodies by, forexample, B-cells; and/or the activation of suppressor T-cells and/or γδT-cells directed specifically to an antigen or antigens present in thecomposition or vaccine of interest. These responses may serve, forexample, to neutralize infectivity, and/or mediate antibody-complement,or antibody dependent cell cytotoxicity (ADCC) to provide protection toan immunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that elicit an immunologicalresponse. The term may be used interchangeably with the term“immunogen.” An “epitope” is that portion of given species (e.g., anantigenic molecule or antigenic complex) that determines itsimmunological specificity. An epitope is within the scope of the presentdefinition of antigen. Commonly, an epitope is a polypeptide orpolysaccharide in a naturally occurring antigen. In artificial antigensit can be a low molecular weight substance such as an arsanilic acidderivative. Normally, a B-cell epitope will include at least about 5amino acids but can be as small as 3-4 amino acids. A T-cell epitope,such as a CTL epitope, will typically include at least about 7-9 aminoacids, and a helper T-cell epitope will typically include at least about12-20 amino acids. The term “antigen” denotes both subunit antigens,i.e., antigens which are separate and discrete from a whole organism orcell with which the antigen is associated in nature, as well as killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes or tumor cells. Antibodies such as anti-idiotype antibodies, orfragments thereof, and synthetic peptide mimotopes, which can mimic anantigen or antigenic determinant, are also captured under the definitionof antigen as used herein. Similarly, an oligonucleotide orpolynucleotide which expresses an antigen or antigenic determinant invivo, such as in gene therapy and DNA immunization applications, is alsoincluded in the definition of antigen herein.

Thus, for purposes of the present invention, antigens can be derivedfrom any of the various viruses, bacteria, parasites, fungi and othermicrobes, as well as any of the various tumor antigens. Antigens alsoinclude nucleic acids which express an antigen or antigenic determinantin vivo. As a few specific examples, antigens may be proteins from orderived from the herpes virus family, including proteins derived fromherpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2glycoproteins gB, gD and gH; proteins derived from cytomegalovirus (CMV)including CMV gB and gH; proteins derived from hepatitis family ofviruses, including hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis Evirus (HEV) and hepatitis G virus (HGV); proteins, including gp120,gp160, gp41, p24gag and p55gag envelope proteins, derived from HIV,including members of the various genetic subtypes of HIV isolatesHIV_(IIIb), HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235),HIV-1_(US4), HIV-2; proteins derived from simian immunodeficiency virus(SN); and proteins derived from Neisseria meningitidis (A, B, C, Y),Hemophilus influenza type B (HIB), Helicobacter pylori; human serumalbumin and ovalbumin, among many others.

An immunogenic composition which contains an antigen in accordance withthe present invention displays “enhanced immunogenicity” when itpossesses a greater capacity to elicit an immune response than theimmune response elicited by an equivalent amount of the antigenadministered using a different delivery system, e.g., wherein theantigen is administered as a soluble protein. Thus, an immunogenic orvaccine composition may display “enhanced immunogenicity” because theantigen is more strongly immunogenic or because a lower dose or fewerdoses of antigen are necessary to achieve an immune response in thesubject to which the antigen is administered. Such enhancedimmunogenicity can be determined by administering the antigencomposition and antigen controls to animals and comparing antibodytiters and/or cellular-mediated immunity against the two using standardassays described herein.

The term “adjuvant” or “immunological adjuvant” refers to any substancethat assists or modifies the action of an antigen in the immune system.Adjuvants can potentiate humoral and/or cellular immunity.

The term “excipient” refers to any essentially accessory substance thatmay be present in the finished dosage form. For example, the term“excipient” includes vehicles, binders, disintegrants, fillers(diluents), suspending/dispersing agents, and so forth.

As used herein, the phrase “vector construct” generally refers to anyassembly that is capable of directing the expression of a nucleic acidsequence(s) or gene(s) of interest. A “DNA vector construct” refers to aDNA molecule that is capable of directing the expression of a nucleicacid sequence(s) or gene(s) of interest. One specific type of DNA vectorconstruct is a plasmid, which is a circular episomal DNA moleculecapable of autonomous replication within a host cell. Typically, aplasmid is a circular double stranded DNA loop into which additional DNAsegments can be ligated. pCMV is one specific plasmid that is well knownin the art. Other DNA vector constructs are known, which are based onRNA viruses. These DNA vector constructs typically comprise a promoterthat functions in a eukaryotic cell, 5′ of a cDNA sequence for which thetranscription product is an RNA vector construct (e.g., an alphavirusRNA vector replicon), and a 3′ termination region. Other examples ofvector constructs include RNA vector constructs (e.g., alphavirus vectorconstructs) and the like. As used herein, “RNA vector construct”, “RNAvector replicon” and “replicon” refer to an RNA molecule that is capableof directing its own amplification or self-replication in vivo,typically within a target cell. The RNA vector construct is useddirectly, without the requirement for introduction of DNA into a celland transport to the nucleus where transcription would occur. By usingthe RNA vector for direct delivery into the cytoplasm of the host cell,autonomous replication and translation of the heterologous nucleic acidsequence occurs efficiently.

The term “homogeneous” refers to a mixture or blend of components thatis generally uniform in structure and composition with littlevariability throughout the mixture. Different portions of a homogeneousmixture exhibit essentially the same physical and chemical propertiessubstantially at every place throughout the mixture. The stoichiometryin a homogeneous mixture is also substantially constant throughout themixture.

The terms “suspension” refers to particles that are dispersed in aliquid.

The term “aqueous suspension” refers to particles that are suspended inan aqueous (continuous) phase liquid that contains water.

A “stable” homogeneous suspension is a suspension in which the size ofthe suspended particles does not substantially change (e.g., change byno more that about 10%) over time.

3. Homogeneous Suspension of Immune-Potentiating Compounds

In one aspect, the invention is a homogeneous suspension comprising (a)a Benzonaphthyridine compound of Formula I or Formula II,pharmaceutically acceptable salt, pharmaceutically acceptable solvate,N-oxide derivative, protected derivative, individual isomer or mixtureof isomers thereof; (b) a surfactant, and (c) a suspension agent. Thehomogeneous suspension contain an aqueous phase, that can be water orany other suitable aqueous phase, such as buffered aqueous solutions.

Preferably, the homogeneous suspension is stable for at least about 2weeks, at least about 4 weeks, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 10 months, at least about 11 months, atleast about 1 year or longer, at 4° C.

The homogeneous suspensions preferably have a size distribution wherein50% of the particles (D50) are smaller than about 10 μM, smaller thanabout 9 μM, smaller than about 8 μM, smaller than about 7 μM, smallerthan about 6 μM, smaller than about 5 μM, smaller than about 4 μM,smaller than about 3 μM, smaller than about 2 μM, smaller than about 1μM, or smaller than about 0.9 μM. Alternatively or in addition, thehomogeneous suspensions can have a size distribution wherein 90% of theparticles (D90) are smaller than about 10 μM, smaller than about 9 μM,smaller than about 8 μM, smaller than about 7 μM, smaller than about 6μM, smaller than about 5 μM, smaller than about 4 μM, smaller than about3 μM, or smaller than about 2 μM.

The homogeneous suspension can contain any suitable surfactant, such asan anionic surfactant, a cationic surfactant, zwitterionic (amphoteric)surfactants, or a non-ionic surfactant. Exemplary anionic surfactantsinclude, e.g., perfluorooctanoate (PFOA or PFO),perfluorooctanesulfonate (PFOS), alkyl sulfate salts such as sodiumdodecyl sulfate (SDS) or ammonium lauryl sulfate, sodium laureth sulfate(also known as sodium lauryl ether sulfate, SLES), alkyl benzenesulfonate, and fatty acid salts. Exemplary cationic surfactants include,e.g., alkyltrimethylammonium salts such as cetyl trimethylammoniumbromide (CTAB, or hexadecyl trimethyl ammonium bromide), cetylpyridiniumchloride (CPC), polyethoxylated tallow amine (POEA), benzalkoniumchloride (BAC), benzethonium chloride (BZT). Exemplary zwitterionic(amphoteric) surfactants include, e.g., dodecyl betaine, cocamidopropylbetaine, and coco ampho glycinate. Exemplary nonionic surfactantsinclude, e.g., alkyl poly(ethylene oxide), alkylphenol poly(ethyleneoxide), copolymers of poly(ethylene oxide) and poly(propylene oxide)(commercially called Poloxamers or Poloxamines), Aayl polyglucosides(e.g., octyl glucoside or decyl maltoside), fatty alcohols (e.g., cetylalcohol or oleyl alcohol), cocamide MEA, cocamide DEA, Pluronic F-68,and Polysorbates, such as Tween 20 (Polysorbate 20), Tween 80(polysorbate 80), and dodecyl dimethylamine oxide. The homogeneoussuspensions can contain two or more surfactants in any combination, ifdesired.

The homogeneous suspensions can contain from about 0.1% to about 10%surfactant (v/v or w/v), about 0.1% to about 5% surfactant, 0.1% toabout 4% surfactant, 0.1% to about 3% surfactant, 0.1% to about 2%surfactant, 0.1% to about 1% surfactant, 0.1% to about 0.5% surfactant,about 0.1% surfactant, about 0.2% surfactant, about 0.3% surfactant,about 0.4% surfactant, about 0.5% surfactant, about 0.6% surfactant,about 0.7% surfactant, about 0.8% surfactant, about 0.9% surfactant, orabout 1% surfactant.

A preferred surfactant for use in the homogeneous suspensions describedherein is Tween 80 (polysorbate 80). Preferrably, the homogeneoussuspension contains about 0.1% to about 5%, 0.1% to about 4%, 0.1% toabout 3%, 0.1% to about 2%, 0.1% to about 1%, 0.1% to about 0.5%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% Tween 80.

The homogeneous suspension can contain any suitable suspension agent,such as a suspension agent that increases viscosity. A variety of suchagents are well-known in the art including, for example, cellulosederivatives (such as carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose), xanthan gum, sodium alginate,carrageenan, tragacanth, cooked starch (e.g., potato starch), guar gum,fumed silica, citronellol, geraniol, dihydro mercinol, linalool, nerol,rhodinal, alphaterpineol, beta-citronellol, rhodinol, citronellanitrile, carvone, fenchone, menthol, and isoborneol. The homogeneoussuspensions can contain two or more suspension agents in anycombination, if desired.

The homogeneous suspensions can contain from about 0.1% to about 10%suspension agent (v/v or w/v), about 0.1% to about 5% suspension agent,0.1% to about 4% suspension agent, 0.1% to about 3% suspension agent,0.1% to about 2% suspension agent, 0.1% to about 1% suspension agent,0.1% to about 0.5% suspension agent, about 0.1% suspension agent, about0.2% suspension agent, about 0.3% suspension agent, about 0.4%suspension agent, about 0.5% suspension agent, about 0.6% suspensionagent, about 0.7% suspension agent, about 0.8% suspension agent, about0.9% suspension agent, or about 1% suspension agent.

A preferred suspension agent for use in the homogeneous suspensionsdescribed herein is carboxymethyl cellulose (CMC). Preferrably, thehomogeneous suspension contains about 0.1% to about 5%, 0.1% to about4%, 0.1% to about 3%, 0.1% to about 2%, 0.1% to about 1%, 0.1% to about0.5%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% carboxymethylcellulose (CMC).

The homogeneous suspensions also contain a Benzonaphthyridine compoundof Formula I or Formula II, pharmaceutically acceptable salt,pharmaceutically acceptable solvate, N-oxide derivative, protectedderivative, individual isomer or mixture of isomers thereof. Thehomogeneous suspensions can contain Benzonaphthyridine compound at aconcentration of from about 0.1 mg/mL to about 50 mg/ml, about 0.1 mg/mLto about 40 mg/ml, about 0.1 mg/mL to about 30 mg/ml, about 0.1 mg/mL toabout 20 mg/ml, about 0.1 mg/mL to about 10 mg/ml, about 0.1 mg/mL toabout 5 mg/ml, about 0.5 mg/mL, about 1.0 mg/mL, about 1.5 mg/ml, about2 mg/ml, about 2.5 mg/ml, about 3 mg/ml, about 3.5 mg/ml, about 4 mg/ml,about 4.5 mg/ml, about 5 mg/ml, about 5.5 mg/ml, about 6 mg/ml, about6.5 mg/ml, about 7 mg/ml, about 7.5 mg/ml, about 8 mg/ml, about 8.5mg/ml, about 9 mg/ml, about 9.5 mg/ml, about 10 mg/ml, about 15 mg/ml,or about 20 mg/mL.

In preferred embodiments, the homogeneous suspensions contain aBenzonaphthyridine compound selected from2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol;2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine; ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate;2-(4-(dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine,and 2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine.

In another preferred embodiments, the homogeneous suspensions contain aBenzonaphthyridine compound selected from2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol;2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine; ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate;2-(4-(dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine,2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine, and2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol.

In particularly preferred embodiments, the homogeneous formulationcomprises 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine.

In another particularly preferred embodiments, the homogeneousformulation comprises2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine,2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine,or2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol.

In certain embodiments, the homogeneous formulation comprises (a) aBenzonaphthyridine compound of Formula I or Formula II, pharmaceuticallyacceptable salt, pharmaceutically acceptable solvate, N-oxidederivative, protected derivative, individual isomer or mixture ofisomers thereof; (b) 0.1% to about 5% Tween 80, and (c) 0.1% to about10% carboxymethyl cellulose, and is stable. In certain examples of theseembodiments, the homogeneous formulation has a D50 of about 1 and,optionally, a D90 of about 2. Preferrably, the homogeneous formulationsare stable for at least about one month. For example, the homogeneousformulations can be stable for at least about one month, at least abouttwo months, at least about three months, at least about four months, atleast about five months, at least about six months, at least about sevenmonths, at least about eight months, at least about nine months, atleast about ten months, at least about eleven months, or at least abouttwelve months in some embodiments.

Benzonaphthyridine Compounds

Benzonaphthyridine compounds suitable for use in the invention includethe compounds of Formula (I) or Formula (II), pharmaceuticallyacceptable salts, pharmaceutically acceptable solvates (e.g. hydrates),the N-oxide derivatives, prodrug derivatives, protected derivatives,individual isomers and mixture of isomers thereof.

In certain embodiments of the immunogenic compositions provided herein,the benzonaphthyridine compound is a compound having the structure ofFormula (I), or a pharmaceutically acceptable salt thereof:

wherein:

-   R⁴ is selected from H, halogen, —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²),    —N(R¹¹R¹²), —N(R⁹)₂, —NHN(R⁹)₂, —SR⁷, —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷,    —LR⁸, —LR¹⁰, —OLR⁸, —OLR¹⁰, C₁-C₆alkyl, C₁-C₆heteroalkyl,    C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy,    C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and    C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl,    C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy,    C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and    C₃-C₈heterocycloalkyl groups of R⁴ are each optionally substituted    with 1 to 3 substituents independently selected from halogen, —CN,    —NO₂, —R⁷, —OR⁸, —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,    —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂, —S(O)₂R⁸,    —S(O)R⁸, —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;-   each L is independently selected from a bond, —(O(CH₂)_(m))_(t)—,    C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene, wherein the    C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene of L are each    optionally substituted with 1 to 4 substituents independently    selected from halogen, —R⁸, —OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,    —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂;-   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,    C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,    C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and    C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl,    C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,    C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl    groups of R⁷ are each optionally substituted with 1 to 3 R¹³ groups;-   each R⁸ is independently selected from H, —CH(R¹⁰)₂, C₁-C₈alkyl,    C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy,    C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,    C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl,    C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,    C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each    optionally substituted with 1 to 3 substituents independently    selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,    —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹², —N(R⁹)₂,    —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹,    —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;-   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,    —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆ heteroalkyl and    C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆alkyl that    together with N they are attached to form a C₃-C₈heterocycloalkyl,    wherein the C₃-C₈heterocycloalkyl ring optionally contains an    additional heteroatom selected from N, O and S, and wherein the    C₁-C₆ alkyl, C₁-C₆ heteroalkyl, C₃-C₆ cycloalkyl, or    C₃-C₈heterocycloalkyl groups of R⁹ are each optionally substituted    with 1 to 3 substituents independently selected from —CN, R¹¹,    —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹, —C(O)OR¹¹, —NR¹¹R¹²,    —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹²,    —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;-   each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl,    C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl,    C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are    optionally substituted with 1 to 3 substituents selected from    halogen, —R⁸, —OR⁸, —LR⁹, —LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸,    —CO₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂;-   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl,    and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl,    C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, and    C₃-C₈heterocycloalkyl groups of R¹¹ and R¹² are each optionally    substituted with 1 to 3 substituents independently selected from    halogen, —CN, R⁸, —OR⁸, —C(O)R⁸, ⁻OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂,    —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸, —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl,    —S(O)₂R⁸, —S(O)₂N(R⁹)₂, —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and    C₁-C₆haloalkoxy;-   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken together    with the N atom to which they are attached form an optionally    substituted C₃-C₈heterocycloalkyl ring optionally containing an    additional heteroatom selected from N, O and S;-   each R¹³ is independently selected from halogen, —CN, —LR⁹, —LOR⁹,    —OLR⁹, —LR¹⁰, —LOR¹⁰, —OLR¹⁰, —LR⁸, —LOR⁸, —OLR⁸, —LSR⁸, —LSR¹⁰,    —LC(O)R⁸, —OLC(O)R⁸, —LC(O)OR⁸, —LC(O)R¹⁰, —LOC(O)OR⁸, —LC(O)NR⁹R¹¹,    —LC(O)NR⁹R⁸, —LN(R⁹)₂, —LNR⁹R⁸, —LNR⁹R¹⁰, —LC(O)N(R⁹)₂, —LS(O)₂R⁸,    —LS(O)R⁸, —LC(O)NR⁸OH, —LNR⁹C(O)R⁸, —LNR⁹C(O)OR⁸, —LS(O)₂N(R⁹)₂,    —OLS(O)₂N(R⁹)₂, —LNR⁹S(O)₂R⁸, —LC(O)NR⁹LN(R⁹)₂, —LP(O)(OR⁸)₂,    —LOP(O)(OR⁸)₂, —LP(O)(OR¹⁰)₂ and —OLP(O)(OR¹⁰)₂;-   each R^(A) is independently selected from —R⁸, —R⁷, —OR⁷, —OR⁸,    —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸,    —NR⁹C(O)N(R⁹)₂, —NR⁹C(S)N(R⁹)₂, —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸,    —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸, —C(O)CH₂C(O)R⁸,    —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸, —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂,    —OC(O)N(R⁹)₂, —OC(O)R⁸, —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸,    —S(O)₃R⁸, —SO₂N(R⁹)₂, —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸,    —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸,    —CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸; or two adjacent    R^(A) substituents on the ring to which they are bonded form a 5-6    membered ring that contains up to two heteroatoms as ring members;-   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7 or 8;-   each m is independently selected from 1, 2, 3, 4, 5 and 6, and-   t is 1, 2, 3, 4, 5, 6, 7 or 8.

In other embodiments, the benzonaphthyridine compound is a compoundhaving the structure of Formula (II), or a pharmaceutically acceptablesalt thereof:

wherein:

-   R⁴ is selected from H, halogen, —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²),    —N(R¹¹R¹²), —N(R⁹)₂, —NHN(R⁹)₂, —SR⁷, —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷,    —LR⁸, LR¹⁰, —OLR⁸, —OLR¹⁰, C₁-C₆alkyl, C₁-C₆heteroalkyl,    C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy,    C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and    C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl,    C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy,    C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and    C₃-C₈heterocycloalkyl groups of R⁴ are each optionally substituted    with 1 to 3 substituents independently selected from halogen, —CN,    —NO₂, —R⁷, —OR⁸, —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,    —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂, —S(O)₂R⁸,    —S(O)R⁸, —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;-   each L is independently selected from a bond, —(O(CH₂)_(m))_(t)—,    C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene, wherein the    C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene of L are each    optionally substituted with 1 to 4 substituents independently    selected from halogen, —R⁸, —OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,    —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂;-   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,    C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,    C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and    C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl,    C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,    C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl    groups of R⁷ are each optionally substituted with 1 to 3 R¹³ groups;-   each R⁸ is independently selected from H, —CH(R¹⁰)₂, C₁-C₈alkyl,    C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy,    C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,    C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl,    C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,    C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each    optionally substituted with 1 to 3 substituents independently    selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,    —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹², —N(R⁹)₂,    —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹,    —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;-   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,    —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆ heteroalkyl and    C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆alkyl that    together with N they are attached to form a C₃-C₈heterocycloalkyl,    wherein the C₃-C₈heterocycloalkyl ring optionally contains an    additional heteroatom selected from N, O and S, and wherein the    C₁-C₆ alkyl, C₁-C₆ heteroalkyl, C₃-C₆ cycloalkyl, or    C₃-C₈heterocycloalkyl groups of R⁹ are each optionally substituted    with 1 to 3 substituents independently selected from —CN, R¹¹,    —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹, —C(O)OR¹¹, —NR¹¹R¹²,    —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹²,    —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;-   each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl,    C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl,    C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are    optionally substituted with 1 to 3 substituents selected from    halogen, —R⁸, —OR⁸, —LR⁹, —LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸,    —CO₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂;-   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl,    and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl,    C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, and    C₃-C₈heterocycloalkyl groups of R¹¹ and R¹² are each optionally    substituted with 1 to 3 substituents independently selected from    halogen, —CN, R⁸, —OR⁸, —C(O)R⁸, ⁻OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂,    —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸, —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl,    —S(O)₂R⁸, —S(O)₂N(R⁹)₂, —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and    C₁-C₆haloalkoxy;-   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken together    with the N atom to which they are attached form an optionally    substituted C₃-C₈heterocycloalkyl ring optionally containing an    additional heteroatom selected from N, O and S;-   each R¹³ is independently selected from halogen, —CN, —LR⁹, —LOR⁹,    —OLR⁹, —LR¹⁰, —LOR¹⁰, —OLR¹⁰, —LR⁸, —LOR⁸, —OLR⁸, —LSR⁸, —LSR¹⁰,    —LC(O)R⁸, —OLC(O)R⁸, —LC(O)OR⁸, —LC(O)R¹⁰, —LOC(O)OR⁸, —LC(O)NR⁹R¹¹,    —LC(O)NR⁹R⁸, —LN(R⁹)₂, —LNR⁹R⁸, —LNR⁹R¹⁰, —LC(O)N(R⁹)₂, —LS(O)₂R⁸,    —LS(O)R⁸, —LC(O)NR⁸OH, —LNR⁹C(O)R⁸, —LNR⁹C(O)OR⁸, —LS(O)₂N(R⁹)₂,    —OLS(O)₂N(R⁹)₂, —LNR⁹S(O)₂R⁸, —LC(O)NR⁹LN(R⁹)₂, —LP(O)(OR⁸)₂,    —LOP(O)(OR⁸)₂, —LP(O)(OR¹⁰)₂ and —OLP(O)(OR¹⁰)₂;-   R^(A) is selected from —R⁸, —R⁷, —OR⁷, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸,    —NO₂, —CN, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂,    —NR⁹C(S)N(R⁹)₂, —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂,    —NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸, —C(O)CH₂C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸,    —C(O)R⁸, —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸,    —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂, —S(O)R⁸,    —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂,    —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸, —CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and    —(CH₂)_(n)NHC(O)R⁸;-   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7 or 8;-   each m is independently selected from 1, 2, 3, 4, 5 and 6, and-   t is 1, 2, 3, 4, 5, 6, 7 or 8.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, each R¹³ is selected from —LR⁹, —LOR⁹, —OLR⁹, —LR¹⁰,—LOR¹⁰, —OLR¹⁰, —LR⁸, —LOR⁸, —OLR⁸, —LSR⁸, —LSR¹⁰, —LC(O)R⁸, —OLC(O)R⁸,—LC(O)OR⁸, —LC(O)R¹⁰, —LOC(O)OR⁸, —LC(O)NR⁹R¹¹, —LC(O)NR⁹R⁸, —LN(R⁹)₂,—LNR⁹R⁸, —LNR⁹R¹⁰, —LC(O)N(R⁹)₂, —LS(O)₂R⁸, —LS(O)R⁸, —LC(O)NR⁸OH,—LNR⁹C(O)R⁸, —LNR⁹C(O)OR⁸, —LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂, —LNR⁹S(O)₂R⁸,—LC(O)NR⁹LN(R⁹)₂, —LP(O)(OR⁸)₂, —LOP(O)(OR⁸)₂, —LP(O)(OR¹⁰)₂ and—OLP(O)(OR¹⁰)₂, and each R^(A) is independently selected from —R⁷, —OR⁷,—R⁸, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —N(R⁹)₂, —S(O)₂R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂,—S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —CH═CHCO₂R⁸, (CH₂)_(n)CO₂R⁸, —NR⁹SO₂R⁸,—P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, each L is independently selected from a—(O(CH₂)_(m))_(t)—, and C₁-C₆alkyl, wherein the C₁-C₆alkyl of L isoptionally substituted with 1 to 4 substituents independently selectedfrom halogen, —R⁸, —OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,—P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, R^(A) is H or C₁-C₆alkyl.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, R^(A) is H, —CH₃ or —CH₂CH₃; and each R¹³ isindependently selected from H, —CH₃, —CH₂CH₃, —CF₃, —CH₂OH, —OCH₃,—COOCH₃, —COOCH₂CH₃, F, Cl, Br, —CH₂OCH₃, CH₂OCH₂CH₃, —N(CH₃)₂,—((O(CH₂)₂)₂₋₄OH, —O(CH₂)₂₋₄—OH, —O(CH₂)₂₋₄—(PO₃H₂), —O(CH₂)₂₋₄—COOH,—O(CH₂)₂₋₄—CH(CH₃)₂ and C₂-C₆ alkyl substituted with 1-3 substituentsselected from —OH, —CH₃, —COOH, —COOCH₃, cyclopropyl, —O(CH₂)₂₋₄—COOH,—O(CH₂)₂₋₄(PO₃H₂), and —COOCH₂CH₃.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, each R⁸ is independently selected from H, —CH(R¹⁰)₂,C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy,C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl,C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy,C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl andC₁-C₆haloalkoxy groups of R⁸ are each optionally substituted with 1 to 3substituents independently selected from —CN, R¹¹, —OR¹¹, —SR¹¹,—C(O)R¹¹, —OC(O)R¹¹, —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰,—NR¹¹R¹², —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,—S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, R⁸ is H or C₁-C₆alkyl.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, R⁹ is H or C₁-C₆alkyl.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, R^(A) is H or —CH₃.

In certain embodiments of the benzonaphthyridine compounds of Formula Ior Formula II, R⁴ is H.

In certain embodiments, the benzonaphthyridine compounds is selectedfrom:

-   2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol;-   2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;-   2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine;-   ethyl    4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate;-   2-(4-(dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine,-   and    2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine.

Benzonaphthyridine compounds of Formula (I) or Formula (II) can beprepared using suitable methods, such as the methods disclosed in WO2009/111337, the entire contents of which are incorporated herein byreference. The person of ordinary skill is directed to WO 2009/111337 atpages 71 to 83 which disclose Schemes I-XIX, which are exemplary schemessuitable for producing compounds of Formula (I) or Formula (II).

The compounds of Formula (I) or Formula (II), pharmaceuticallyacceptable salts, solvates, N-oxides, prodrugs and isomers thereof, andpharmaceutical compositions provided herein also includes all suitableisotopic variations of such compounds, and pharmaceutically acceptablesalts, solvates, N-oxides, prodrugs and isomers thereof, andpharmaceutical compositions. An isotopic variation of a compoundprovided herein or a pharmaceutically acceptable salt thereof is definedas one in which at least one atom is replaced by an atom having the sameatomic number but an atomic mass different from the atomic mass usuallyfound in nature. Examples of isotopes that may be incorporated into thecompounds provided herein and pharmaceutically acceptable salts thereofinclude but are not limited to isotopes of hydrogen, carbon, nitrogenand oxygen such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁵S, ¹⁸F, ³⁶Cland ¹²³I. Certain isotopic variations of the compounds provided hereinand pharmaceutically acceptable salts thereof, for example, those inwhich a radioactive isotope such as ³H or ¹⁴C is incorporated, areuseful in drug and/or substrate tissue distribution studies. Inparticular examples, ³H and ¹⁴C isotopes may be used for their ease ofpreparation and detectability. In other examples, substitution withisotopes such as ²H may afford certain therapeutic advantages resultingfrom greater metabolic stability, such as increased in vivo half-life orreduced dosage requirements. Isotopic variations of the compounds, andpharmaceutically acceptable salts, solvates, N-oxides, prodrugs andisomers thereof, and pharmaceutical compositions provided herein areprepared by conventional procedures using appropriate isotopicvariations of suitable reagents.

4. Immune Potentiation and Immunogenic Compositions

The homogeneous suspension of SMIPs, i.e. compounds of Formula I orFormula II, described herein can be used as immune potentiators.Compounds of Formula I or Formula II contain a hydrophobic core, and thehomogeneous suspensions provide the advantage of allowing an effectiveamount of SMIP to be conveniently administered to a subject in needthereof.

In one aspect, the invention is a method for potentiation an immuneresponse, comprising administering an effective amount of a homogeneoussuspension of the invention to a subject in need thereof. The immuneresponse can be a naturally occurring immune response, or an inducedimmune response, for example induced by immunization. When thehomogeneous suspension is administered to potentiate an induced immuneresponse, it is preferred that it is administer at substantially thesame time as the agent that induces the immune response. For example, aneffective amount of a homogeneous suspension can be administeredconcurrently with an immunogenic composition or vaccine, or administeredwithin a period of about 1 day before or after the immunogeniccomposition or vaccine is administered.

In another aspect, the invention provides an immunogenic compositioncomprising an antigen and a homogeneous suspension of a SMIP, i.e.compounds of Formula I or Formula II. In certain embodiments, theimmunogenic composition further comprises another immunostimulatoryagent, such as an adjuvant. The immunogenic composition can contain anydesired antigen and/or adjuvant. For example, the immunogeniccomposition can contain a bacterial antigen, bacterial vesicle antigen,viral antigen, fungal antigen, protozoan antigen, plant antigen, STDantigen, respiratory antigen, pediatric vaccine antigen, antigensuitable for elderly or immunocompromized individuals, antigen suitablefor use in adolescent vaccines, or tumor antigen, as described herein.The immunogenic composition can also contain an adjuvant (e.g., alum,MF59) as described herein.

In certain embodiments, the immunogenic composition contains an amountof homogeneous composition that is sufficient to enhance an immuneresponse to the antigen. Such immunogenic compositions can be used toproduce vaccines. In certain embodiments, the vaccines are prophylactic(i.e. to prevent infection), while in other embodiments, such vaccinesare therapeutic (i.e. to treat infection).

The immunostimulatory effect referred to herein is often an enhancementof the immunogenic composition's effect. In certain embodiments theenhancement of the efficacy of the immunogenic composition is by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 100%, relative tothe effect of the immunogenic composition in the absence of thehomogeneous suspension of SMIP.

In certain embodiments, the enhancement of the immunogenic composition'seffect is measured by the increased effectiveness of the immunogeniccomposition for achieving its protective effects. In certainembodiments, this increased effectiveness is measured as a decreasedprobability that a subject receiving the immunogenic composition willexperience a condition for which the immunogenic composition isconsidered protective, or a decrease in duration or severity of theeffects of such condition. In other embodiments, this increasedeffectiveness is measured as an increase in a titer of an antibodyelicited by the immunogenic composition in a treated subject.

The immunogenic compositions may further comprise a pharmaceuticallyacceptable carrier. Such carriers are include, but are not limited to,proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, sucrose, trehalose,lactose, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. The immunogenic compositions typically alsocontain diluents, such as water, saline, and glycerol, and optionallycontain other excipients, such as wetting or emulsifying agents, and pHbuffering substances.

(a) Adjuvants

In certain embodiments, immunogenic compositions optionally include oneor more immunoregulatory agents. In certain embodiments, one or more ofthe immunoregulatory agents include one or more adjuvants. Suchadjuvants include, but are not limited to, a TH1 adjuvant and/or a TH2adjuvant, further discussed below. In certain embodiments, the adjuvantsused in immunogenic compositions provide herein include, but are notlimited to:

1. Mineral-Containing Compositions;

2. Oil Emulsions;

3. Saponin Formulations;

4. Virosomes and Virus-Like Particles;

5. Bacterial or Microbial Derivatives;

6. Human Immunomodulators;

7. Bioadhesives and Mucoadhesives;

8. Microparticles;

9. Liposomes;

10. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations;

11. Polyphosphazene (PCPP);

12. Muramyl Peptides, and

13. Imidazoquinolone Compounds.

Mineral-containing compositions suitable for use as adjuvants include,but are not limited to, mineral salts, such as aluminium salts andcalcium salts. By way of example only, such mineral salts include,hydroxides (e.g. oxyhydroxides, including aluminium hydroxides andaluminium oxyhydroxides), phosphates (e.g. hydroxyphosphates andorthophosphates, including aluminium phosphates, aluminiumhydroxyphosphates, aluminium orthophosphates and calcium phosphate),sulfates (e.g. aluminium sulfate), or mixtures of different mineralcompounds. Such mineral salts are in any suitable form, such as, by wayof example only, gel, crystalline, and amorphous forms. In certainembodiments, such mineral containing compositions are formulated as aparticle of the metal salt. In certain embodiments, components of theimmunogenic compositions described herein are adsorbed to such mineralsalts. In certain embodiments, an aluminium hydroxide and/or aluminiumphosphate adjuvant is used in the immunogenic compositions describedherein. In other embodiments, antigens used in an immunogeniccomposition described herein are adsorbed to such aluminium hydroxideand/or aluminium phosphate adjuvants. In certain embodiments, a calciumphosphate adjuvant is used in the immunogenic compositions describedherein. In other embodiments, antigens used in an immunogeniccomposition described herein are adsorbed to such calcium phosphateadjuvants.

In certain embodiments, aluminum phosphates are used as an adjuvant inthe immunogenic compositions described herein. In other embodiments,aluminum phosphates are used as an adjuvant in the immunogeniccompositions described herein, wherein such compositions include a H.influenzae disaccharide antigen. In certain embodiments, the adjuvant isamorphous aluminium hydroxyphosphate with a PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. In other embodiments,adsorption with a low dose of aluminium phosphate is used, by way ofexample only, between 50 and 100 μg Al³⁺ per conjugate per dose. Wherethere is more than one conjugate in a composition, not all conjugatesneed to be adsorbed.

Oil emulsions suitable for use as adjuvants include, but are not limitedto, squalene-water emulsions (such as MF59 (5% Squalene, 0.5% Tween 80,and 0.5% Span 85, formulated into submicron particles using amicrofluidizer), Complete Freund's adjuvant (CFA) and incompleteFreund's adjuvant (IFA).

Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin formulations suitablefor use as adjuvants include, but are not limited to, saponins from thebark of the Quillaia saponaria Molina tree, from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). In certain embodiments, saponin formulationssuitable for use as adjuvants include, but are not limited to, purifiedformulations including, but are not limited to, QS7, QS17, QS18, QS21,QH-A, QH-B and QH-C. QS21 is marketed as STIMULOM™. In otherembodiments, saponin formulations include sterols, cholesterols andlipid formulations, such as unique particles formed by the combinationsof saponins and cholesterols called immunostimulating complexes(ISCOMs). In certain embodiments, the ISCOMs also include a phospholipidsuch as phosphatidylethanolamine or phosphatidylcholine. Any knownsaponin can be used in ISCOMs. In certain embodiments, the ISCOMincludes one or more of QuilA, QHA & QHC. In other embodiments, theISCOMS are optionally devoid of an additional detergent.

Virosomes and virus-like particles (VLPs) suitable for use as adjuvantsinclude, but are not limited to, one or more proteins from a virusoptionally combined or formulated with a phospholipid. Such virosomesand VLPs are generally non-pathogenic, non-replicating and generally donot contain any of the native viral genome. In certain embodiments, theviral proteins are recombinantly produced, while in other embodimentsthe viral proteins are isolated from whole viruses.

The viral proteins suitable for use in virosomes or VLPs include, butare not limited to, proteins derived from influenza virus (such as HA orNA), Hepatitis B virus (such as core or capsid proteins), Hepatitis Evirus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Diseasevirus, Retrovirus, Norwalk virus, human Papilloma virus, HIV,RNA-phages, Q□-phage (such as coat proteins), GA-phage, fr-phage, AP205phage, and Ty (such as retrotransposon Ty protein p1).

Bacterial or microbial derivatives suitable for use as adjuvantsinclude, but are not limited to, bacterial or microbial derivatives suchas non-toxic derivatives of enterobacterial lipopolysaccharide (LPS),Lipid A derivatives, immunostimulatory oligonucleotides andADP-ribosylating toxins and detoxified derivatives thereof. Suchnon-toxic derivatives of LPS include, but are not limited to,monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is amixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6acylated chains. Other non-toxic LPS derivatives include monophosphoryllipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives(e.g. RC-529). Lipid A derivatives include, but are not limited to,derivatives of lipid A from Escherichia coli (e.g. OM-174).

Immunostimulatory oligonucleotides used as adjuvants include, but arenot limited to, nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Such CpG sequences can bedouble-stranded or single-stranded. In certain embodiments, suchnucleotide sequences are double-stranded RNAs or oligonucleotidescontaining palindromic or poly(dG) sequences. In other embodiments, theCpG's include nucleotide modifications/analogs such as phosphorothioatemodifications.

In certain embodiments the CpG sequence are directed to TLR9, and incertain embodiments the motif is GTCGTT or TTCGTT. In certainembodiments the CpG sequence is specific for inducing a Th1 immuneresponse, such as, by way of example only, a CpG-A ODN, or in otherembodiments the CpG sequence is more specific for inducing a B cellresponse, such as, by way of example only, a CpG-B ODN. In certainembodiments the CpG is a CpG-A ODN.

In certain embodiments the CpG oligonucleotide is constructed so thatthe 5′ end is accessible for receptor recognition. In other embodimentstwo CpG oligonucleotide sequences are optionally attached at their 3′ends to form “immunomers”.

A particularly useful adjuvant based around immunostimulatoryoligonucleotides is known as IC-31™. In certain embodiments, an adjuvantused with immunogenic compositions described herein, includes a mixtureof (i) an oligonucleotide (such as, by way of example only, between15-40 nucleotides) including at least one (and preferably multiple) CpImotifs (such as, by way of example only, a cytosine linked to an inosineto form a dinucleotide), and (ii) a polycationic polymer, such as, byway of example only, an oligopeptide (such as, by way of example only,between 5-20 amino acids) including at least one (and preferablymultiple) Lys-Arg-Lys tripeptide sequence(s). In certain embodiments,the oligonucleotide is a deoxynucleotide comprising 26-mer sequence5′-(IC)₁₃-3′. In other embodiments, the polycationic polymer is apeptide comprising 11-mer amino acid sequence KLKLLLLLKLK.

In certain embodiments, bacterial ADP-ribosylating toxins and detoxifiedderivatives thereof are used as adjuvants in the immunogeniccompositions described herein. In certain embodiments, such proteins arederived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). In other embodiments, the toxin or toxoidis in the form of a holotoxin, comprising both A and B subunits. Inother embodiments, the A subunit contains a detoxifying mutation;whereas the B subunit is not mutated. In other embodiments, the adjuvantis a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192.

The human immunomodulators suitable for use as adjuvants include, butare not limited to, cytokines, such as, by way of example only,interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferons(such as, by way of example only, interferon-□), macrophage colonystimulating factor, and tumor necrosis factor.

The bioadhesives and mucoadhesives used as adjuvants in the immunogeniccompositions described herein include, but are not limited to,esterified hyaluronic acid microspheres, and cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. In certain embodiments,chitosan and derivatives thereof are used as in the vaccine compositionsdescribed herein adjuvants.

The microparticles suitable for use as adjuvants include, but are notlimited to, microparticles formed from materials that are biodegradableand non-toxic (e.g. a poly(.alpha.-hydroxy acid), a polyhydroxybutyricacid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide). In certain embodiments, such microparticlesare treated to have a negatively-charged surface (e.g. with SDS) or apositively-charged surface (e.g. with a cationic detergent, such asCTAB). The microparticles suitable for use as adjuvants have a particlediameter of about 100 nm to about 150 μm in diameter. In certainembodiments, the particle diameter is about 200 nm to about 30 μm, andin other embodiments the particle diameter is about 500 nm to 10 μm.

The polyoxyethylene ether and polyoxyethylene ester formulationssuitable for use as adjuvants include, but are not limited to,polyoxyethylene sorbitan ester surfactants in combination with anoctoxynol, and polyoxyethylene alkyl ethers or ester surfactants incombination with at least one additional non-ionic surfactant such as anoctoxynol. In certain embodiments, the polyoxyethylene ethers areselected from polyoxyethylene-9-lauryl ether (laureth 9),polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

The muramyl peptides suitable for use as adjuvants include, but are notlimited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

In certain embodiments, one or more compounds of Formula (I) used as animmune potentiator are included in compositions having combinations ofone or more of the adjuvants identified above. Such combinationsinclude, but are not limited to,

-   -   (1) a saponin and an oil-in-water emulsion;    -   (2) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g.        3dMPL);    -   (3) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g.        3dMPL)+a cholesterol;    -   (4) a saponin (e.g. QS21)+3dMPTL+IL-12 (optionally including a        sterol);    -   (5) combinations of 3dMPL with, for example, QS21 and/or        oil-in-water emulsions;    -   (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5%        pluronic-block polymer L121, and thr-MDP, either microfluidized        into a submicron emulsion or vortexed to generate a larger        particle size emulsion.    -   (7) RIBI™ adjuvant system (RAS), (Ribi Immunochem) containing 2%        squalene, 0.2% Tween 80, and one or more bacterial cell wall        components from the group consisting of monophosphorylipid A        (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),        preferably MPL+CWS (Detox™); and    -   (8) one or more mineral salts (such as an aluminum salt)+a        non-toxic derivative of LPS (such as 3dMPL).

In other embodiments, the adjuvant combinations used in the immunogeniccombinations provided herein include combinations of Th1 and Th2adjuvants such as, by way of example only, CpG and alum or resiquimodand alum.

In certain embodiments, the immunogenic compositions provided hereinelicit both a cell mediated immune response as well as a humoral immuneresponse. In other embodiments, the immune response induces long lasting(e.g. neutralising) antibodies and a cell mediated immunity that quicklyresponds upon exposure to the infectious agent.

TH1 adjuvants can be used to elicit a TH1 immune response. A TH1adjuvant will generally elicit increased levels of IgG2a productionrelative to immunization of the antigen without adjuvant. TH1 adjuvantssuitable for use in immunogenic compositions provided herein include,but are not limited to, saponin formulations, virosomes and virus likeparticles, non-toxic derivatives of enterobacterial lipopolysaccharide(LPS), immunostimulatory oligonucleotides. In certain embodiments, theimmunostimulatory oligonucleotides used as TH1 adjuvants in theimmunogenic compositions provided herein contain a CpG motif.

TH2 adjuvants can be used to elicit a TH2 immune response. A TH2adjuvant will generally elicit increased levels of IgG1 productionrelative to immunization of the antigen without adjuvant. TH2 adjuvantssuitable for use in immunogenic compositions provided herein include,but are not limited to, mineral containing compositions, oil-emulsions,and ADP-ribosylating toxins and detoxified derivatives thereof. Incertain embodiments, the mineral containing compositions used as TH2adjuvants in the immunogenic compositions provided herein are aluminiumsalts.

In certain embodiments, the immunogenic compositions provided hereininclude a TH1 adjuvant and a TH2 adjuvant. In other embodiments, suchcompositions elicit an enhanced TH1 and an enhanced TH2 response, suchas, an increase in the production of both IgG1 and IgG2a productionrelative to immunization without an adjuvant. In still otherembodiments, such compositions comprising a combination of a TH1 and aTH2 adjuvant elicit an increased TH1 and/or an increased TH2 immuneresponse relative to immunization with a single adjuvant (i.e.; relativeto immunization with a TH1 adjuvant alone or immunization with a TH2adjuvant alone).

In certain embodiments, the immune response is one or both of a TH1immune response and a TH2 response. In other embodiments, the immuneresponse provides for one or both of an enhanced TH1 response and anenhanced TH2 response.

In certain embodiments, the enhanced immune response is one or both of asystemic and a mucosal immune response. In other embodiments, the immuneresponse provides for one or both of an enhanced systemic and anenhanced mucosal immune response. In certain embodiments, the mucosalimmune response is a TH2 immune response. In certain embodiments, themucosal immune response includes an increase in the production of IgA.

(b) Antigens

In certain embodiments, the immunogenic composition comprises at leastone antigen, which may be a bacterial antigen or a cancer-associatedantigen, or a viral antigen. In certain embodiments, the homogeneoussuspensions provided herein are included in prophylactic vaccines orused in combination with prophylactic vaccines. In certain embodiments,the homogeneous suspensions provided herein are included in, or are usedin combination with, therapeutic viral vaccines. In certain embodiments,the homogeneous suspensions provided herein are included in, or are usedin combination with, with cancer vaccines.

Antigens for use with the immunogenic compositions provided hereininclude, but are not limited to, one or more of the following antigensset forth below, or antigens derived from one or more of the pathogensset forth below.

Bacterial Antigens

Bacterial antigens suitable for use in immunogenic compositions providedherein include, but are not limited to, proteins, polysaccharides,lipopolysaccharides, and outer membrane vesicles which are isolated,purified or derived from a bacteria. In certain embodiments, thebacterial antigens include bacterial lysates and inactivated bacteriaformulations. In certain embodiments, the bacterial antigens areproduced by recombinant expression. In certain embodiments, thebacterial antigens include epitopes which are exposed on the surface ofthe bacteria during at least one stage of its life cycle. Bacterialantigens are preferably conserved across multiple serotypes. In certainembodiments, the bacterial antigens include antigens derived from one ormore of the bacteria set forth below as well as the specific antigensexamples identified below:

Neisseria meningitidis: Meningitidis antigens include, but are notlimited to, proteins, saccharides (including a polysaccharide,oligosaccharide, lipooligosaccharide or lipopolysaccharide), orouter-membrane vesicles purified or derived from N. meningitidesserogroup such as A, C, W135, Y, X and/or B. In certain embodimentsmeningitides protein antigens are be selected from adhesions,autotransporters, toxins, Fe acquisition proteins, and membraneassociated proteins (preferably integral outer membrane protein).

Streptococcus pneumoniae: Streptococcus pneumoniae antigens include, butare not limited to, a saccharide (including a polysaccharide or anoligosaccharide) and/or protein from Streptococcus pneumoniae. Thesaccharide may be a polysaccharide having the size that arises duringpurification of the saccharide from bacteria, or it may be anoligosaccharide achieved by fragmentation of such a polysaccharide. Inthe 7-valent PREVNAR™ product, for instance, 6 of the saccharides arepresented as intact polysaccharides while one (the 18C serotype) ispresented as an oligosaccharide. In certain embodiments saccharideantigens are selected from one or more of the following pneumococcalserotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,17F, 18C, 19A, 19F, 20, 22F, 23F, and/or 33F. An immunogenic compositionmay include multiple serotypes e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes. 7-valent,9-valent, 10-valent, 11-valent and 13-valent conjugate combinations arealready known in the art, as is a 23-valent unconjugated combination.For example, an 10-valent combination may include saccharide fromserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valentcombination may further include saccharide from serotype 3. A 12-valentcombination may add to the 10-valent mixture: serotypes 6A and 19A; 6Aand 22F; 19A and 22F; 6A and 15B; 19A and 15B; r 22F and 15B; A13-valent combination may add to the 11-valent mixture: serotypes 19Aand 22F; 8 and 12F; 8 and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12Fand 19A; 12F and 22F; 15B and 19A; 15B and 22F. etc. In certainembodiments, protein antigens may be selected from a protein identifiedin WO98/18931, WO98/18930, U.S. Pat. No. 6,699,703, U.S. Pat. No.6,800,744, WO97/43303, WO97/37026, WO 02/079241, WO 02/34773, WO00/06737, WO 00/06738, WO 00/58475, WO 2003/082183, WO 00/37105, WO02/22167, WO 02/22168, WO 2003/104272, WO 02/08426, WO 01/12219, WO99/53940, WO 01/81380, WO 2004/092209, WO 00/76540, WO 2007/116322,LeMieux et al., Infect. Imm. (2006) 74:2453-2456, Hoskins et al., J.Bacteriol. (2001) 183:5709-5717, Adamou et al., Infect. Immun. (2001)69(2):949-958, Briles et al., J. Infect. Dis. (2000) 182:1694-1701,Talkington et al., Microb. Pathog. (1996) 21(1):17-22, Bethe et al.,FEMS Microbiol. Lett. (2001) 205(1):99-104, Brown et al., Infect. Immun.(2001) 69:6702-6706, Whalen et al., FEMS Immunol. Med. Microbiol. (2005)43:73-80, Jomaa et al., Vaccine (2006) 24(24):5133-5139. In otherembodiments, Streptococcus pneumoniae proteins may be selected from thePoly Histidine Triad family (PhtX), the Choline Binding Protein family(CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytXtruncate chimeric proteins, pneumolysin (Ply), PspA, PsaA, Sp128, SpIO1,Sp130, Sp125, Sp133, pneumococcal pilus subunits.

Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcusantigens include, but are not limited to, a protein identified in WO02/34771 or WO 2005/032582 (including GAS 40), fusions of fragments ofGAS M proteins (including those described in WO 02/094851, and Dale,Vaccine (1999) 17:193-200, and Dale, Vaccine 14(10): 944-948),fibronectin binding protein (Sfbl), Streptococcal heme-associatedprotein (Shp), and Streptolysin S (SagA).

Moraxella catarrhalis: Moraxella antigens include, but are not limitedto, antigens identified in WO 02/18595 and WO 99/58562, outer membraneprotein antigens (HMW-OMP), C-antigen, and/or LPS.

Bordetella pertussis: Pertussis antigens include, but are not limitedto, pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) fromB. pertussis, optionally also combination with pertactin and/oragglutinogens 2 and 3.

Burkholderia: Burkholderia antigens include, but are not limited toBurkholderia mallei, Burkholderia pseudomallei and Burkholderia cepacia.

Staphylococcus aureus: Staph aureus antigens include, but are notlimited to, a polysaccharide and/or protein from S. aureus. S. aureuspolysaccharides include, but are not limited to, type 5 and type 8capsular polysaccharides (CP5 and CP8) optionally conjugated to nontoxicrecombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, type336 polysaccharides (336PS), polysaccharide intercellular adhesions(PIA, also known as PNAG). S. aureus proteins include, but are notlimited to, antigens derived from surface proteins, invasins(leukocidin, kinases, hyaluronidase), surface factors that inhibitphagocytic engulfment (capsule, Protein A), carotenoids, catalaseproduction, Protein A, coagulase, clotting factor, and/ormembrane-damaging toxins (optionally detoxified) that lyse eukaryoticcell membranes (hemolysins, leukotoxin, leukocidin). In certainembodiments, S. aureus antigens may be selected from a proteinidentified in WO 02/094868, WO 2008/019162, WO 02/059148, WO 02/102829,WO 03/011899, WO 2005/079315, WO 02/077183, WO 99/27109, WO 01/70955, WO00/12689, WO 00/12131, WO 2006/032475, WO 2006/032472, WO 2006/032500,WO 2007/113222, WO 2007/113223, WO 2007/113224. In other embodiments, S.aureus antigens may be selected from IsdA, IsdB, IsdC, SdrC, SdrD, SdrE,ClfA, ClfB, SasF, SasD, SasH (AdsA), Spa, EsaC, EsxA, EsxB, Emp,HlaH35L, CP5, CP8, PNAG, 336PS.

Staphylococcus epidermis: S. epidermidis antigens include, but are notlimited to, slime-associated antigen (SAA).

Clostridium tetani (Tetanus): Tetanus antigens include, but are notlimited to, tetanus toxoid (TT). In certain embodiments such antigensare used as a carrier protein in conjunction/conjugated with theimmunogenic compositions provided herein.

Clostridium perfringens: Antigens include, but are not limited to,Epsilon toxin from Clostridium perfringen.

Clostridium botulinums (Botulism): Botulism antigens include, but arenot limited to, those derived from C. botulinum.

Cornynebacterium diphtheriae (Diphtheria): Diphtheria antigens include,but are not limited to, diphtheria toxin, preferably detoxified, such asCRM₁₉₇. Additionally antigens capable of modulating, inhibiting orassociated with ADP ribosylation are contemplated forcombination/co-administration/conjugation with the immunogeniccompositions provided herein. In certain embodiments, the diphtheriatoxoids are used as carrier proteins.

Haemophilus influenzae B (Hib): Hib antigens include, but are notlimited to, a Hib saccharide antigen.

Pseudomonas aeruginosa: Pseudomonas antigens include, but are notlimited to, endotoxin A, Wzz protein, P. aeruginosa LPS, LPS isolatedfrom PAO1 (O5 serotype), and/or Outer Membrane Proteins, including OuterMembrane Proteins F (OprF).

Legionella pneumophila. Bacterial antigens derived from Legionellapneumophila.

Coxiella burnetii. Bacterial antigens derived from Coxiella bumetii.

Brucella. Bacterial antigens derived from Brucella, including but notlimited to, B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis,B. suis and B. pinnipediae.

Francisella. Bacterial antigens derived from Francisella, including butnot limited to, F. novicida, F. philomiragia and F. tularensis.

Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcusantigens include, but are not limited to, a protein or saccharideantigen identified in WO 02/34771, WO 03/093306, WO 04/041157, or WO2005/002619 (including proteins GBS 80, GBS 104, GBS 276 and GBS 322,and including saccharide antigens derived from serotypes Ia, Ib, Ia/c,II, III, IV, V, VI, VII and VIII).

Neiserria gonorrhoeae: Gonorrhoeae antigens include, but are not limitedto, Por (or porin) protein, such as PorB (see Zhu et al., Vaccine (2004)22:660-669), a transferring binding protein, such as TbpA and TbpB (SeePrice et al., Infection and Immunity (2004) 71(1):277-283), a opacityprotein (such as Opa), a reduction-modifiable protein (Rmp), and outermembrane vesicle (OMV) preparations (see Plante et al, J InfectiousDisease (2000) 182:848-855), also see, e.g., WO99/24578, WO99/36544,WO99/57280, WO02/079243).

Chlamydia trachomatis: Chlamydia trachomatis antigens include, but arenot limited to, antigens derived from serotypes A, B, Ba and C (agentsof trachoma, a cause of blindness), serotypes L₁, L₂ & L₃ (associatedwith Lymphogranuloma venereum), and serotypes, D-K. In certainembodiments, chlamydia trachomas antigens include, but are not limitedto, an antigen identified in WO 00/37494, WO 03/049762, WO 03/068811, orWO 05/002619, including PepA (CT045), LcrE (CT089), ArtJ (CT381), DnaK(CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA (CT444), AtosS(CT467), CT547, Eno (CT587), HrtA (CT823), and MurG (CT761).

Treponema pallidum (Syphilis): Syphilis antigens include, but are notlimited to, TmpA antigen.

Haemophilus ducreyi (causing chancroid): Ducreyi antigens include, butare not limited to, outer membrane protein (DsrA).

Enterococcus faecalis or Enterococcus faecium: Antigens include, but arenot limited to, a trisaccharide repeat or other Enterococcus derivedantigens.

Helicobacter pylori: H pylori antigens include, but are not limited to,Cag, Vac, Nap, HopX, HopY and/or urease antigen.

Staphylococcus saprophyticus: Antigens include, but are not limited to,the 160 kDa hemagglutinin of S. saprophyticus antigen.

Yersinia enterocolitica Antigens include, but are not limited to, LPS.

E. coli: E. coli antigens may be derived from enterotoxigenic E. coli(ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli(DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E.coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC antigensinclude, but are not limited to, accessory colonization factor(orf3526), orf353, bacterial Ig-like domain (group 1) protein (orf405),orf1364, NodT-family outer-membrane-factor-lipoprotein effluxtransporter (orf1767), gspK (orf3515), gspJ (orf3516), tonB-dependentsiderophore receptor (orf3597), fimbrial protein (orf3613), upec-948,upec-1232, A chain precursor of the type-1 fimbrial protein (upec-1875),yap H homolog (upec-2820), and homolysin A (recp-3768).

Bacillus anthracis (anthrax): B. anthracis antigens include, but are notlimited to, A-components (lethal factor (LF) and edema factor (EF)),both of which can share a common B-component known as protective antigen(PA). In certain embodiments, B. anthracis antigens are optionallydetoxified.

Yersinia pestis (plague): Plague antigens include, but are not limitedto, F1 capsular antigen, LPS, Yersinia pestis V antigen.

Mycobacterium tuberculosis: Tuberculosis antigens include, but are notlimited to, lipoproteins, LPS, BCG antigens, a fusion protein of antigen85B (Ag85B), ESAT-6 optionally formulated in cationic lipid vesicles,Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associatedantigens, and MPT51 antigens.

Rickettsia: Antigens include, but are not limited to, outer membraneproteins, including the outer membrane protein A and/or B (OmpB), LPS,and surface protein antigen (SPA).

Listeria monocytogenes: Bacterial antigens include, but are not limitedto, those derived from Listeria monocytogenes.

Chlamydia pneumoniae: Antigens include, but are not limited to, thoseidentified in WO 02/02606.

Vibrio cholerae: Antigens include, but are not limited to, proteinaseantigens, LPS, particularly lipopolysaccharides of Vibrio cholerae II,O1 Inaba O-specific polysaccharides, V. cholera O139, antigens of IEM108vaccine and Zonula occludens toxin (Zot).

Salmonella typhi (typhoid fever): Antigens include, but are not limitedto, capsular polysaccharides preferably conjugates (Vi, i.e. vax-TyVi).

Borrelia burgdorferi (Lyme disease): Antigens include, but are notlimited to, lipoproteins (such as OspA, OspB, Osp C and Osp D), othersurface proteins such as OspE-related proteins (Erps), decorin-bindingproteins (such as DbpA), and antigenically variable VI proteins, such asantigens associated with P39 and P13 (an integral membrane protein, VISEAntigenic Variation Protein.

Porphyromonas gingivalis: Antigens include, but are not limited to, P.gingivalis outer membrane protein (OMP).

Klebsiella: Antigens include, but are not limited to, an OMP, includingOMP A, or a polysaccharide optionally conjugated to tetanus toxoid.

Other bacterial antigens used in the immunogenic compositions providedherein include, but are not limited to, capsular antigens,polysaccharide antigens or protein antigens of any of the above. Otherbacterial antigens used in the immunogenic compositions provided hereininclude, but are not limited to, an outer membrane vesicle (OMV)preparation. Additionally, Other bacterial antigens used in theimmunogenic compositions provided herein include, but are not limitedto, live, attenuated, and/or purified versions of any of theaforementioned bacteria. In certain embodiments, the bacterial antigensused in the immunogenic compositions provided herein are derived fromgram-negative, while in other embodiments they are derived fromgram-positive bacteria. In certain embodiments, the bacterial antigensused in the immunogenic compositions provided herein are derived fromaerobic bacteria, while in other embodiments they are derived fromanaerobic bacteria.

In certain embodiments, any of the above bacterial-derived saccharides(polysaccharides, LPS, LOS or oligosaccharides) are conjugated toanother agent or antigen, such as a carrier protein (for exampleCRM₁₉₇). In certain embodiments, such conjugations are directconjugations effected by reductive amination of carbonyl moieties on thesaccharide to amino groups on the protein. In other embodiments, thesaccharides are conjugated through a linker, such as, with succinamideor other linkages provided in Bioconjugate Techniques, 1996 and CRC,Chemistry of Protein Conjugation and Cross-Linking, 1993.

In certain embodiments useful for the treatment or prevention ofNeisseria infection and related diseases and disorders, recombinantproteins from N. meningitidis for use in the immunogenic compositionsprovided herein may be found in WO99/24578, WO99/36544, WO99/57280,WO00/22430, WO96/29412, WO01/64920, WO03/020756, WO2004/048404, andWO2004/032958. Such antigens may be used alone or in combinations. Wheremultiple purified proteins are combined then it is helpful to use amixture of 10 or fewer (e.g. 9, 8, 7, 6, 5, 4, 3, 2) purified antigens.

A particularly useful combination of antigens for use in the immunogeniccompositions provided herein is disclosed in Giuliani et al. (2006) ProcNatl Acad Sci USA 103(29):10834-9 and WO2004/032958, and so animmunogenic composition may include 1, 2, 3, 4 or 5 of (1) a ‘NadA’protein (aka GNA1994 and NMB1994); (2) a ‘fHBP’ protein (aka ‘741’,LP2086, GNA1870, and NMB1870); (3) a ‘936’ protein (aka GNA2091 andNMB2091); (4) a ‘953’ protein (aka GNA1030 and NMB1030); and (5) a ‘287’protein (aka GNA2132 and NMB2132). Other possible antigen combinationsmay comprise a transferrin binding protein (e.g. TbpA and/or TbpB) andan Hsf antigen. Other possible purified antigens for use in theimmunogenic compositions provided herein include proteins comprising oneof the following amino acid sequences: SEQ ID NO:650 from WO99/24578;SEQ ID NO:878 from WO99/24578; SEQ ID NO:884 from WO99/24578; SEQ IDNO:4 from WO99/36544; SEQ ID NO:598 from WO99/57280; SEQ ID NO:818 fromWO99/57280; SEQ ID NO:864 from WO99/57280; SEQ ID NO:866 fromWO99/57280; SEQ ID NO:1196 from WO99/57280; SEQ ID NO:1272 fromWO99/57280; SEQ ID NO:1274 from WO99/57280; SEQ ID NO:1640 fromWO99/57280; SEQ ID NO:1788 from WO99/57280; SEQ ID NO:2288 fromWO99/57280; SEQ ID NO:2466 from WO99/57280; SEQ ID NO:2554 fromWO99/57280; SEQ ID NO:2576 from WO99/57280; SEQ ID NO:2606 fromWO99/57280; SEQ ID NO:2608 from WO99/57280; SEQ ID NO:2616 fromWO99/57280; SEQ ID NO:2668 from WO99/57280; SEQ ID NO:2780 fromWO99/57280; SEQ ID NO:2932 from WO99/57280; SEQ ID NO:2958 fromWO99/57280; SEQ ID NO:2970 from WO99/57280; SEQ ID NO:2988 fromWO99/57280 (each of the forgoing amino acid sequences is herebyincorporated by reference from the cited document), or a polypeptidecomprising an amino acid sequence which: (a) has 50% or more identity(e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) to said sequences; and/or(b) comprises a fragment of at least n consecutive amino acids from saidsequences, wherein n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferredfragments for (b) comprise an epitope from the relevant sequence. Morethan one (e.g., 2, 3, 4, 5, 6) of these polypeptides may be included inthe immunogenic compositions.

The fHBP antigen falls into three distinct variants (WO2004/048404). AnN. meningitidis serogroup vaccine based upon the immunogeniccompositions disclosed herein utilizing one of the compounds disclosedherein may include a single fHBP variant, but is will usefully includean fHBP from each of two or all three variants. Thus the immunogeniccomposition may include a combination of two or three different purifiedfHBPs, selected from: (a) a first protein, comprising an amino acidsequence having at least a % sequence identity to SEQ ID NO: 1 and/orcomprising an amino acid sequence consisting of a fragment of at least xcontiguous amino acids from SEQ ID NO: 1; (b) a second protein,comprising an amino acid sequence having at least b % sequence identityto SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of afragment of at least y contiguous amino acids from SEQ ID NO: 2; and/or(c) a third protein, comprising an amino acid sequence having at least c% sequence identity to SEQ ID NO: 3 and/or comprising an amino acidsequence consisting of a fragment of at least z contiguous amino acidsfrom SEQ ID NO: 3

SEQ ID NO: 1 VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ SEQ ID NO: 2VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ SEQ ID NO: 3VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGK Q.

The value of a is at least 85, e.g., 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, or more. The value of b is at least 85, e.g.,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.The value of c is at least 85, e.g., 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, or more. The values of a, b and c are notintrinsically related to each other.

The value of x is at least 7, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of yis at least 7, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 225, 250). The value of z is at least 7,e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 225, 250). The values of x, y and z are not intrinsicallyrelated to each other.

In some embodiments, the immunogenic compositions as disclosed hereinwill include fHBP protein(s) that are lipidated, e.g., at a N-terminalcysteine. In other embodiments they will not be lipidated

A useful immunogenic composition as disclosed herein includes purifiedproteins comprises a mixture of: (i) a first polypeptide having aminoacid sequence SEQ ID NO: 4; (ii) a second polypeptide having amino acidsequence SEQ ID NO: 5; and (iii) a third polypeptide having amino acidsequence SEQ ID NO: 6. See Giuliani et al. (2006) Proc Natl Acad Sci USA103(29):10834-9 and WO2004/032958. A useful immunogenic composition asdisclosed herein includes purified proteins comprises a mixture of: (i)a first polypeptide having at least a % sequence identity to amino acidsequence SEQ ID NO: 4; (ii) a second polypeptide having at least b %sequence identity to amino acid sequence SEQ ID NO: 5; and (iii) a thirdpolypeptide having at least a % sequence identity to amino acid sequenceSEQ ID NO: 6.

SEQ ID NO: 4 MASPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGGQDMAAVSEENTGNGGAAATDKPKNEDEGAQNDMPQNAADTDSLTPNHTPASNMPAGNMENQAPDAGESEQPANQPDMANTADGMQGDDPSAGGENAGNTAAQGTNQAENNQTAGSQNPASSTNPSATNSGGDFGRTNVGNSVVIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDGKNDKFVGLVADSVQMKGINQYIIFYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPSKGEMLAGTAVYNGEVLHFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGDGLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGSGGGGATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDI QIEAAKQ SEQ ID NO: 5MVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGTSPATQARVKTVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRGSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ SEQ ID NO: 6ATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADHDTRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVG.

Bacterial Vesicle Antigens

The immunogenic compositions as disclosed herein may include outermembrane vesicles. Such outer membrane vesicles may be obtained from awide array of pathogenic bacteria and used as antigenic components ofthe immunogenic compositions as disclosed herein. Vesicles for use asantigenic components of such immunogenic compositions include anyproteoliposomic vesicle obtained by disrupting a bacterial outermembrane to form vesicles therefrom that include protein components ofthe outer membrane. Thus the term includes OMVs (sometimes referred toas ‘blebs’), microvesicles (MVs, see, e.g., WO02/09643) and ‘nativeOMVs’ (‘NOMVs’ see, e.g., Katial et al. (2002) Infect. Immun.70:702-707). Immnogenic compositions as disclosed herein that includevesicles from one or more pathogenic bacteria can be used in thetreatment or prevention of infection by such pathogenic bacteria andrelated diseases and disorders.

MVs and NOMVs are naturally-occurring membrane vesicles that formspontaneously during bacterial growth and are released into culturemedium. MVs can be obtained by culturing bacteria such as Neisseria inbroth culture medium, separating whole cells from the smaller MVs in thebroth culture medium (e.g., by filtration or by low-speed centrifugationto pellet only the cells and not the smaller vesicles), and thencollecting the MVs from the cell-depleted medium (e.g., by filtration,by differential precipitation or aggregation of MVs, by high-speedcentrifugation to pellet the MVs). Strains for use in production of MVscan generally be selected on the basis of the amount of MVs produced inculture (see, e.g., U.S. Pat. No. 6,180,111 and WO01/34642 describingNeisseria with high MV production).

OMVs are prepared artificially from bacteria, and may be prepared usingdetergent treatment (e.g., with deoxycholate), or by non detergent means(see, e.g., WO04/019977). Methods for obtaining suitable OMVpreparations are well known in the art. Techniques for forming OMVsinclude treating bacteria with a bile acid salt detergent (e.g., saltsof lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid,deoxycholic acid, cholic acid, ursocholic acid, etc., with sodiumdeoxycholate (EP0011243 and Fredriksen et al. (1991) NIPH Ann.14(2):67-80) being preferred for treating Neisseria) at a pHsufficiently high not to precipitate the detergent (see, e.g.,WO01/91788). Other techniques may be performed substantially in theabsence of detergent (see, e.g., WO04/019977) using techniques such assonication, homogenisation, microfluidisation, cavitation, osmoticshock, grinding, French press, blending, etc. Methods using no or lowdetergent can retain useful antigens such as NspA in Neisserial OMVs.Thus a method may use an OMV extraction buffer with about 0.5%deoxycholate or lower, e.g., about 0.2%, about 0.1%, <0.05% or zero.

A useful process for OMV preparation is described in WO05/004908 andinvolves ultrafiltration on crude OMVs, rather than instead of highspeed centrifugation. The process may involve a step ofultracentrifugation after the ultrafiltration takes place.

Vesicles can be prepared from any pathogenic strain such as Neisseriaminigtidis for use with the invention. Vesicles from Neisserialmeningitidis serogroup B may be of any serotype (e.g., 1, 2a, 2b, 4, 14,15, 16, etc.), any serosubtype, and any immunotype (e.g., L1; L2; L3;L3,3,7; L10; etc.). The meningococci may be from any suitable lineage,including hyperinvasive and hypervirulent lineages, e.g., any of thefollowing seven hypervirulent lineages: subgroup I; subgroup III;subgroup IV 1; ET 5 complex; ET 37 complex; A4 cluster; lineage 3. Theselineages have been defined by multilocus enzyme electrophoresis (MLEE),but multilocus sequence typing (MLST) has also been used to classifymeningococci, e.g., the ET 37 complex is the ST 11 complex by MLST, theET 5 complex is ST-32 (ET-5), lineage 3 is ST 41/44, etc. Vesicles canbe prepared from strains having one of the following subtypes: P1.2;P1.2,5; P1.4; P1.5; P1.5,2; P1.5,c; P1.5c, 10; P1.7,16; P1.7,16b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13; P1.13; P1.14; P1.21,16; P1.22,14.

Vesicles included in the immunogenic compositions disclosed herein maybe prepared from wild type pathogenic strains such as N. meningitidisstrains or from mutant strains. By way of example, WO98/56901 disclosespreparations of vesicles obtained from N. meningitidis with a modifiedfur gene. WO02/09746 teaches that nspA expression should be up regulatedwith concomitant porA and cps knockout. Further knockout mutants of N.meningitidis for OMV production are disclosed in WO02/0974, WO02/062378,and WO04/014417. WO06/081259 discloses vesicles in which fHBP isupregulated. Claassen et al. (1996) 14(10):1001-8, disclose theconstruction of vesicles from strains modified to express six differentPorA subtypes. Mutant Neisseria with low endotoxin levels, achieved byknockout of enzymes involved in LPS biosynthesis, may also be used (see,e.g., WO99/10497 and Steeghs et al. (2001) i20:6937-6945). These orothers mutants can all be used with the invention.

Thus N. meningitidis serogroup B strains included in the immunogeniccompositions disclosed herein may in some embodiments express more thanone PorA subtype. Six valent and nine valent PorA strains havepreviously been constructed. The strain may express 2, 3, 4, 5, 6, 7, 8or 9 of PorA subtypes: P1.7,16; P1.5-1,2-2; P1,19,15-1; P1.5-2,10; P1.121,13; P1.7-2,4; P1.22,14; P1.7-1,1 and/or P1.18-1,3,6. In otherembodiments a strain may have been down regulated for PorA expression,e.g., in which the amount of PorA has been reduced by at least 20%(e.g., >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, etc.), or evenknocked out, relative to wild type levels (e.g., relative to strainH44/76, as disclosed in WO03/105890).

In some embodiments N. meningitidis serogroup B strains may over express(relative to the corresponding wild-type strain) certain proteins. Forinstance, strains may over express NspA, protein 287 (WO01/52885—alsoreferred to as NMB2132 and GNA2132), one or more fHBP (WO06/081259 andU.S. Pat. Pub. 2008/0248065—also referred to as protein 741, NMB1870 andGNA1870), TbpA and/or TbpB (WO00/25811), Cu,Zn-superoxide dismutase(WO00/25811), etc.

In some embodiments N. meningitidis serogroup B strains may include oneor more of the knockout and/or over expression mutations. Preferredgenes for down regulation and/or knockout include: (a) Cps, CtrA, CtrB,CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC,PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB (WO01/09350); (b) CtrA,CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc,PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB(WO02/09746); (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB,Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB(WO02/062378); and (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB,SiaD, SynA, SynB, and/or SynC (WO04/014417).

Where a mutant strain is used, in some embodiments it may have one ormore, or all, of the following characteristics: (i) down regulated orknocked-out LgtB and/or GalE to truncate the meningococcal LOS; (ii) upregulated TbpA; (iii) up regulated Hsf; (iv) up regulated Omp85; (v) upregulated LbpA; (vi) up regulated NspA; (vii) knocked-out PorA; (viii)down regulated or knocked-out FrpB; (ix) down regulated or knocked-outOpa; (x) down regulated or knocked-out Opc; (xii) deleted cps genecomplex. A truncated LOS can be one that does not include asialyl-lacto-N-neotetraose epitope, e.g., it might be agalactose-deficient LOS. The LOS may have no α chain.

If LOS is present in a vesicle then it is possible to treat the vesicleso as to link its LOS and protein components (“intra-bleb” conjugation(WO04/014417)).

The immunogenic compositions as disclosed herein may include mixtures ofvesicles from different strains. By way of example, WO03/105890discloses vaccine comprising multivalent meningococcal vesiclecompositions, comprising a first vesicle derived from a meningococcalstrain with a serosubtype prevalent in a country of use, and a secondvesicle derived from a strain that need not have a serosubtype preventin a country of use. WO06/024946 discloses useful combinations ofdifferent vesicles. A combination of vesicles from strains in each ofthe L2 and L3 immunotypes may be used in some embodiments.

Vesicle-based antigens can be prepared from N. meningitidis serogroupsother than serogroup B (e.g., WO01/91788 discloses a process forserogroup A). The immunogenic compositions disclosed herein accordinglycan include vesicles prepared serogroups other than B (e.g. A, C, W135and/or Y) and from bacterial pathogens other than Neisseria.

Viral Antigens

Viral antigens suitable for use in the immunogenic compositions providedherein include, but are not limited to, inactivated (or killed) virus,attenuated virus, split virus formulations, purified subunitformulations, viral proteins which may be isolated, purified or derivedfrom a virus, and Virus Like Particles (VLPs). In certain embodiments,viral antigens are derived from viruses propagated on cell culture orother substrate. In other embodiments, viral antigens are expressedrecombinantly. In certain embodiments, viral antigens preferably includeepitopes which are exposed on the surface of the virus during at leastone stage of its life cycle. Viral antigens are preferably conservedacross multiple serotypes or isolates. Viral antigens suitable for usein the immunogenic compositions provided herein include, but are notlimited to, antigens derived from one or more of the viruses set forthbelow as well as the specific antigens examples identified below.

Orthomyxovirus: Viral antigens include, but are not limited to, thosederived from an Orthomyxovirus, such as Influenza A, B and C. In certainembodiments, orthomyxovirus antigens are selected from one or more ofthe viral proteins, including hemagglutinin (HA), neuraminidase (NA),nucleoprotein (NP), matrix protein (M1), membrane protein (M2), one ormore of the transcriptase components (PB1, PB2 and PA). In certainembodiments the viral antigen include HA and NA. In certain embodiments,the influenza antigens are derived from interpandemic (annual) flustrains, while in other embodiments, the influenza antigens are derivedfrom strains with the potential to cause pandemic a pandemic outbreak(i.e., influenza strains with new haemagglutinin compared to thehaemagglutinin in currently circulating strains, or influenza strainswhich are pathogenic in avian subjects and have the potential to betransmitted horizontally in the human population, or influenza strainswhich are pathogenic to humans).

Paramyxoviridae viruses: Viral antigens include, but are not limited to,those derived from Paramyxoviridae viruses, such as Pneumoviruses (RSV),Paramyxoviruses (PIV), Metapneumovirus and Morbilliviruses (Measles).

Pneumovirus: Viral antigens include, but are not limited to, thosederived from a Pneumovirus, such as Respiratory syncytial virus (RSV),Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkeyrhinotracheitis virus. Preferably, the Pneumovirus is RSV. In certainembodiments, pneumovirus antigens are selected from one or more of thefollowing proteins, including surface proteins Fusion (F), Glycoprotein(G) and Small Hydrophobic protein (SH), matrix proteins M and M2,nucleocapsid proteins N, P and L and nonstructural proteins NS1 and NS2.In other embodiments, pneumovirus antigens include F, G and M. Incertain embodiments, pneumovirus antigens are also formulated in orderived from chimeric viruses, such as, by way of example only, chimericRSV/PIV viruses comprising components of both RSV and PIV.

Paramyxovirus: Viral antigens include, but are not limited to, thosederived from a Paramyxovirus, such as Parainfluenza virus types 1-4(PIV), Mumps, Sendai viruses, Simian virus 5, Bovine parainfluenzavirus, Nipahvirus, Henipavirus and Newcastle disease virus. In certainembodiments, the Paramyxovirus is PIV or Mumps. In certain embodiments,paramyxovirus antigens are selected from one or more of the followingproteins: Hemagglutinin-Neuraminidase (HN), Fusion proteins F1 and F2,Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrixprotein (M). In other embodiments, paramyxovirus proteins include HN, F1and F2. In certain embodiments, paramyxovirus antigens are alsoformulated in or derived from chimeric viruses, such as, by way ofexample only, chimeric RSV/PIV viruses comprising components of both RSVand PIV. Commercially available mumps vaccines include live attenuatedmumps virus, in either a monovalent form or in combination with measlesand rubella vaccines (MMR). In other embodiments, the Paramyxovirus isNipahvirus or Henipavirus and the antigens are selected from one or moreof the following proteins: Fusion (F) protein, Glycoprotein (G) protein,Matrix (M) protein, Nucleocapsid (N) protein, Large (L) protein andPhosphoprotein (P).

Poxyiridae: Viral antigens include, but are not limited to, thosederived from Orthopoxvirus such as Variola vera, including but notlimited to, Variola major and Variola minor.

Metapneumovirus: Viral antigens include, but are not limited to,Metapneumovirus, such as human metapneumovirus (hMPV) and avianmetapneumoviruses (aMPV). In certain embodiments, metapneumovirusantigens are selected from one or more of the following proteins,including surface proteins Fusion (F), Glycoprotein (G) and SmallHydrophobic protein (SH), matrix proteins M and M2, nucleocapsidproteins N, P and L. In other embodiments, metapneumovirus antigensinclude F, G and M. In certain embodiments, metapneumovirus antigens arealso formulated in or derived from chimeric viruses.

Morbillivirus: Viral antigens include, but are not limited to, thosederived from a Morbillivirus, such as Measles. In certain embodiments,morbillivirus antigens are selected from one or more of the followingproteins: hemagglutinin (H), Glycoprotein (G), Fusion factor (F), Largeprotein (L), Nucleoprotein (NP), Polymerase phosphoprotein (P), andMatrix (M). Commercially available measles vaccines include liveattenuated measles virus, typically in combination with mumps andrubella (MMR).

Picornavirus: Viral antigens include, but are not limited to, thosederived from Picornaviruses, such as Enteroviruses, Rhinoviruses,Heparnavirus, Cardioviruses and Aphthoviruses. In certain embodiments,the antigens are derived from Enteroviruses, while in other embodimentsthe enterovirus is Poliovirus. In still other embodiments, the antigensare derived from Rhinoviruses. In certain embodiments, the antigens areformulated into virus-like particles (VLPs).

Enterovirus: Viral antigens include, but are not limited to, thosederived from an Enterovirus, such as Poliovirus types 1, 2 or 3,Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6,Echovirus (ECHO) virus) types 1 to 9, 11 to 27 and 29 to 34 andEnterovirus 68 to 71. In certain embodiments, the antigens are derivedfrom Enteroviruses, while in other embodiments the enterovirus isPoliovirus. In certain embodiments, the enterovirus antigens areselected from one or more of the following Capsid proteins VP0, VP1,VP2, VP3 and VP4. Commercially available polio vaccines includeInactivated Polio Vaccine (IPV) and Oral poliovirus vaccine (OPV). Incertain embodiments, the antigens are formulated into virus-likeparticles.

Bunyavirus: Viral antigens include, but are not limited to, thosederived from an Orthohunyavirus, such as California encephalitis virus,a Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such asCrimean-Congo hemorrhagic fever virus.

Rhinovirus: Viral antigens include, but are not limited to, thosederived from rhinovirus. In certain embodiments, the rhinovirus antigensare selected from one or more of the following Capsid proteins: VP0,VP1, VP2, VP2 and VP4. In certain embodiments, the antigens areformulated into virus-like particles (VLPs).

Heparnavirus: Viral antigens include, but are not limited to, thosederived from a Heparnavirus, such as, by way of example only, HepatitisA virus (HAV). Commercially available HAV vaccines include inactivatedHAV vaccine.

Togavirus: Viral antigens include, but are not limited to, those derivedfrom a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus.In certain embodiments, the antigens are derived from Rubivirus, such asby way of example only, Rubella virus. In certain embodiments, thetogavirus antigens are selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3or NSP-4. In certain embodiments, the togavirus antigens are selectedfrom E1, E2 or E3. Commercially available Rubella vaccines include alive cold-adapted virus, typically in combination with mumps and measlesvaccines (MMR).

Flavivirus: Viral antigens include, but are not limited to, thosederived from a Flavivirus, such as Tick-borne encephalitis (TBE) virus,Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japaneseencephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus,St. Louis encephalitis virus, Russian spring-summer encephalitis virus,Powassan encephalitis virus. In certain embodiments, the flavivirusantigens are selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a,NS4b, and NS5. In certain embodiments, the flavivirus antigens areselected from PrM, M and E. Commercially available TBE vaccine includesinactivated virus vaccines. In certain embodiments, the antigens areformulated into virus-like particles (VLPs).

Pestivirus: Viral antigens include, but are not limited to, thosederived from a Pestivirus, such as Bovine viral diarrhea (BVDV),Classical swine fever (CSFV) or Border disease (BDV).

Hepadnavirus: Viral antigens include, but are not limited to, thosederived from a Hepadnavirus, such as Hepatitis B virus. In certainembodiments, the hepadnavirus antigens are selected from surfaceantigens (L, M and S), core antigens (HBc, HBe). Commercially availableHBV vaccines include subunit vaccines comprising the surface antigen Sprotein.

Hepatitis C virus: Viral antigens include, but are not limited to, thosederived from a Hepatitis C virus (HCV). In certain embodiments, the HCVantigens are selected from one or more of E1, E2, E1/E2, NS345polyprotein, NS 345-core polyprotein, core, and/or peptides from thenonstructural regions. In certain embodiments, the Hepatitis C virusantigens include one or more of the following: HCV E1 and or E2proteins, E1/E2 heterodimer complexes, core proteins and non-structuralproteins, or fragments of these antigens, wherein the non-structuralproteins can optionally be modified to remove enzymatic activity butretain immunogenicity. In certain embodiments, the antigens areformulated into virus-like particles (VLPs).

Rhabdovirus: Viral antigens include, but are not limited to, thosederived from a Rhabdovirus, such as a Lyssavirus (Rabies virus) andVesiculovirus (VSV). Rhabdovirus antigens may be selected fromglycoprotein (G), nucleoprotein (N), large protein (L), nonstructuralproteins (NS). Commercially available Rabies virus vaccine comprisekilled virus grown on human diploid cells or fetal rhesus lung cells.

Caliciviridae; Viral antigens include, but are not limited to, thosederived from Calciviridae, such as Norwalk virus, and Norwalk-likeViruses, such as Hawaii Virus and Snow Mountain Virus. In certainembodiments, the antigens are formulated into virus-like particles(VLPs).

Coronavirus: Viral antigens include, but are not limited to, thosederived from a Coronavirus, SARS, Human respiratory coronavirus, Avianinfectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcinetransmissible gastroenteritis virus (TGEV). In certain embodiments, thecoronavirus antigens are selected from spike (S), envelope (E), matrix(M), nucleocapsid (N), and Hemagglutinin-esterase glycoprotein (HE). Incertain embodiments, the coronavirus antigen is derived from a SARSvirus. In certain embodiments, the coronavirus is derived from a SARSviral antigen as described in WO 04/92360.

Retrovirus: Viral antigens include, but are not limited to, thosederived from a Retrovirus, such as an Oncovirus, a Lentivirus or aSpumavirus. In certain embodiments, the oncovirus antigens are derivedfrom HTLV-1, HTLV-2 or HTLV-5. In certain embodiments, the lentivirusantigens are derived from HIV-1 or HIV-2. In certain embodiments, theantigens are derived from HIV-1 subtypes (or clades), including, but notlimited to, HIV-1 subtypes (or clades) A, B, C, D, F, G, H, J. K, O. Inother embodiments, the antigens are derived from HIV-1 circulatingrecombinant forms (CRFs), including, but not limited to, A/B, A/E, A/G,A/G/I, etc. In certain embodiments, the retrovirus antigens are selectedfrom gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. Incertain embodiments, the HIV antigens are selected from gag (p24gag andp55gag), env (gp160 and gp41), pol, tat, nef, rev vpu, miniproteins,(preferably p55 gag and gp140v delete). In certain embodiments, the HIVantigens are derived from one or more of the following strains:HIV_(IIIb), HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235),HIV-1_(US4), HIV-1_(SF162), HIV-1_(TV1), HTV-1_(MJ4). In certainembodiments, the antigens are derived from endogenous humanretroviruses, including, but not limited to, HERV-K (“old” HERV-K and“new” HERV-K).

Reovirus: Viral antigens include, but are not limited to, those derivedfrom a Reovirus, such as an Orthoreovirus, a Rotavirus, an Orbivirus, ora Coltivirus. In certain embodiments, the reovirus antigens are selectedfrom structural proteins λ1, λ2, λ3, μ1, μ2, σ1, σ2, or σ3, ornonstructural proteins σNS, μNS, or σ1s. In certain embodiments, thereovirus antigens are derived from a Rotavirus. In certain embodiments,the rotavirus antigens are selected from VP1, VP2, VP3, VP4 (or thecleaved product VP5 and VP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, orNSP5. In certain embodiments, the rotavirus antigens include VP4 (or thecleaved product VP5 and VP8), and VP7.

Parvovirus: Viral antigens include, but are not limited to, thosederived from a Parvovirus, such as Parvovirus B 19. In certainembodiments, the Parvovirus antigens are selected from VP-1, VP-2, VP-3,NS-1 and NS-2. In certain embodiments, the Parvovirus antigen is capsidprotein VP1 or VP-2. In certain embodiments, the antigens are formulatedinto virus-like particles (VLPs).

Delta hepatitis virus (HDV): Viral antigens include, but are not limitedto, those derived from HDV, particularly δ-antigen from HDV.

Hepatitis E virus (HEV): Viral antigens include, but are not limited to,those derived from HEV.

Hepatitis G virus (HGV): Viral antigens include, but are not limited to,those derived from HGV.

Human Herpesvirus: Viral antigens include, but are not limited to, thosederived from a Human Herpesvirus, such as, by way of example only,Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein-Barrvirus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), HumanHerpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). In certainembodiments, the Human Herpesvirus antigens are selected from immediateearly proteins (α), early proteins (β), and late proteins (γ). Incertain embodiments, the HSV antigens are derived from HSV-1 or HSV-2strains. In certain embodiments, the HSV antigens are selected fromglycoproteins gB, gC, gD and gH, fusion protein (gB), or immune escapeproteins (gC, gE, or gI). In certain embodiments, the VZV antigens areselected from core, nucleocapsid, tegument, or envelope proteins. A liveattenuated VZV vaccine is commercially available. In certainembodiments, the EBV antigens are selected from early antigen (EA)proteins, viral capsid antigen (VCA), and glycoproteins of the membraneantigen (MA). In certain embodiments, the CMV antigens are selected fromcapsid proteins, envelope glycoproteins (such as gB and gH), andtegument proteins. In other embodiments, CMV antigens may be selectedfrom one or more of the following proteins: pp 65, IE1, gB, gD, gH, gL,gM, gN, gO, UL128, UL129, gUL130, UL150, UL131, UL33, UL78, US27, US28,RL5A, RL6, RL10, RL11, RL12, RL13, UL1, UL2, UL4, UL5, UL6, UL7, UL8,UL9, UL10, UL11, UL14, UL15A, UL16, UL17, UL18, UL22A, UL38, UL40,UL41A, UL42, UL116, UL119, UL120, UL121, UL124, UL132, UL147A, UL148,UL142, UL144, UL141, UL140, UL135, UL136, UL138, UL139, UL133, UL135,UL148A, UL148B, UL148C, UL148D, US2, US3, US6, US7, USB, US9, US10,US11, US12, US13, US14, US15, US16, US17, US18, US19, US20, US21, US29,US30 and US34A. CMV antigens may also be fusions of one or more CMVproteins, such as, by way of example only, pp 65/IE1 (Reap et al.,Vaccine (2007) 25:7441-7449). In certain embodiments, the antigens areformulated into virus-like particles (VLPs).

Papovaviruses: Antigens include, but are not limited to, those derivedfrom Papovaviruses, such as Papillomaviruses and Polyomaviruses. Incertain embodiments, the Papillomaviruses include HPV serotypes 1, 2, 4,5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 and65. In certain embodiments, the HPV antigens are derived from serotypes6, 11, 16 or 18. In certain embodiments, the HPV antigens are selectedfrom capsid proteins (L1) and (L2), or E1-E7, or fusions thereof. Incertain embodiments, the HPV antigens are formulated into virus-likeparticles (VLPs). In certain embodiments, the Polyomyavirus virusesinclude BK virus and JK virus. In certain embodiments, the Polyomavirusantigens are selected from VP1, VP2 or VP3.

Adenovirus: Antigens include those derived from Adenovirus. In certainembodiments, the Adenovirus antigens are derived from Adenovirusserotype 36 (Ad-36). In certain embodiments, the antigen is derived froma protein or peptide sequence encoding an Ad-36 coat protein or fragmentthereof (WO 2007/120362).

Further provided are antigens, compositions, methods, and microbesincluded in Vaccines, 4^(th) Edition (Plotkin and Orenstein ed. 2004);Medical Microbiology 4^(th) Edition (Murray et al. ed. 2002); Virology,3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition(B. N. Fields and D. M. Knipe, eds. 1991), which are contemplated inconjunction with the immunogenic compositions provided herein.

Fungal Antigens

Fungal antigens for use in the immunogenic compositions provided hereininclude, but are not limited to, those derived from one or more of thefungi set forth below.

Fungal antigens are derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme.

Fungal pathogens are derived from Aspergillus fumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septataintestinalis and Enterocytozoon bieneusi; the less common are Brachiolaspp, Microsporidium spp., Nosema spp., Pleistophora spp.,Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis,Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale,Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe,Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii,Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaeaspp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolusspp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp,Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp,Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp,Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, andCladosporium spp.

In certain embodiments, the process for producing a fungal antigenincludes a method wherein a solubilized fraction extracted and separatedfrom an insoluble fraction obtainable from fungal cells of which cellwall has been substantially removed or at least partially removed,characterized in that the process comprises the steps of: obtainingliving fungal cells; obtaining fungal cells of which cell wall has beensubstantially removed or at least partially removed; bursting the fungalcells of which cell wall has been substantially removed or at leastpartially removed; obtaining an insoluble fraction; and extracting andseparating a solubilized fraction from the insoluble fraction.

Protazoan Antigens/Pathogens

Protazoan antigens/pathogens for use in the immunogenic compositionsprovided herein include, but are not limited to, those derived from oneor more of the following protozoa: Entamoeba histolytica, Giardialambli, Cryptosporidium parvum, Cyclospora cayatanensis and Toxoplasma.

Plant Antigens/Pathogens

Plan antigens/pathogens for use in the immunogenic compositions providedherein include, but are not limited to, those derived from Ricinuscommunis.

STD Antigens

In certain embodiments, the immunogenic compositions provided hereininclude one or more antigens derived from a sexually transmitted disease(STD). In certain embodiments, such antigens provide for prophylactisfor STD's such as chlamydia, genital herpes, hepatitis (such as HCV),genital warts, gonorrhea, syphilis and/or chancroid. In otherembodiments, such antigens provide for therapy for STD's such aschlamydia, genital herpes, hepatitis (such as HCV), genital warts,gonorrhea, syphilis and/or chancroid. Such antigens are derived from oneor more viral or bacterial STD's. In certain embodiments, the viral STDantigens are derived from HIV, herpes simplex virus (HSV-1 and HSV-2),human papillomavirus (HPV), and hepatitis (HCV). In certain embodiments,the bacterial STD antigens are derived from Neiserria gonorrhoeae,Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, E. coli,and Streptococcus agalactiae. Examples of specific antigens derived fromthese pathogens are described above.

Respiratory Antigens

In certain embodiments, the immunogenic compositions provided hereininclude one or more antigens derived from a pathogen which causesrespiratory disease. By way of example only, such respiratory antigensare derived from a respiratory virus such as Orthomyxoviruses(influenza), Pneumovirus (RSV), Paramyxovirus (Ply), Morbillivirus(measles), Togavirus (Rubella), VZV, and Coronavirus (SARS). In certainembodiments, the respiratory antigens are derived from a bacteria whichcauses respiratory disease, such as, by way of example only,Streptococcus pneumoniae, Pseudomonas aeruginosa, Bordetella pertussis,Mycobacterium tuberculosis, Mycoplasma pneumoniae, Chlamydia pneumoniae,Bacillus anthracis, and Moraxella catarrhalis. Examples of specificantigens derived from these pathogens are described above.

Pediatric Vaccine Antigens

In certain embodiments, the immunogenic compositions provided hereininclude one or more antigens suitable for use in pediatric subjects.Pediatric subjects are typically less than about 3 years old, or lessthan about 2 years old, or less than about 1 years old. Pediatricantigens are administered multiple times over the course of 6 months, 1,2 or 3 years. Pediatric antigens are derived from a virus which maytarget pediatric populations and/or a virus from which pediatricpopulations are susceptible to infection. Pediatric viral antigensinclude, but are not limited to, antigens derived from one or more ofOrthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV andMumps), Morbillivirus (measles), Togavirus (Rubella), Enterovirus(polio), HBV, Coronavirus (SARS), and Varicella-zoster virus (VZV),Epstein Barr virus (EBV). Pediatric bacterial antigens include antigensderived from one or more of Streptococcus pneumoniae, Neisseriameningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridiumtetani (Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilusinfluenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae(Group B Streptococcus), and E. coli. Examples of specific antigensderived from these pathogens are described above.

Antigens Suitable for Use in Elderly or Immunocompromised Individuals

In certain embodiments, the immunogenic compositions provided hereininclude one or more antigens suitable for use in elderly orimmunocompromised individuals. Such individuals may need to bevaccinated more frequently, with higher doses or with adjuvantedformulations to improve their immune response to the targeted antigens.Antigens which are targeted for use in Elderly or Immunocompromisedindividuals include antigens derived from one or more of the followingpathogens: Neisseria meningitides, Streptococcus pneumoniae,Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis,Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis,Clostridium tetani (Tetanus), Cornynebacterium diphtherias (Diphtheria),Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Legionellapneumophila, Streptococcus agalactiae (Group B Streptococcus),Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae,Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV andMumps), Morbillivirus (measles), Togavirus (Rubella), Enterovirus HBV,Coronavirus (SARS), Varicella-zoster virus (VZV), Epstein Barr virus(EBV), Cytomegalovirus (CMV). Examples of specific antigens derived fromthese pathogens are described above.

Antigens Suitable for Use in Adolescent Vaccines

In certain embodiments, the immunogenic compositions provided hereininclude one or more antigens suitable for use in adolescent subjects.Adolescents are in need of a boost of a previously administeredpediatric antigen. Pediatric antigens which are suitable for use inadolescents are described above. In addition, adolescents are targetedto receive antigens derived from an STD pathogen in order to ensureprotective or therapeutic immunity before the beginning of sexualactivity. STD antigens which are suitable for use in adolescents aredescribed above.

Tumor Antigens

In certain embodiments, a tumor antigen or cancer antigen is used inconjunction with the immunogenic compositions provided herein. Incertain embodiments, the tumor antigens is a peptide-containing tumorantigens, such as a polypeptide tumor antigen or glycoprotein tumorantigens. In certain embodiments, the tumor antigen is asaccharide-containing tumor antigen, such as a glycolipid tumor antigenor a ganglioside tumor antigen. In certain embodiments, the tumorantigen is a polynucleotide-containing tumor antigen that expresses apolypeptide-containing tumor antigen, for instance, an RNA vectorconstruct or a DNA vector construct, such as plasmid DNA.

Tumor antigens appropriate for the use in conjunction with theimmunogenic compositions provided herein encompass a wide variety ofmolecules, such as (a) polypeptide-containing tumor antigens, includingpolypeptides (which can range, for example, from 8-20 amino acids inlength, although lengths outside this range are also common),lipopolypeptides and glycoproteins, (b) saccharide-containing tumorantigens, including polysaccharides, mucins, gangliosides, glycolipidsand glycoproteins, and (c) polynucleotides that express antigenicpolypeptides.

In certain embodiments, the tumor antigens are, for example, (a) fulllength molecules associated with cancer cells, (b) homologs and modifiedforms of the same, including molecules with deleted, added and/orsubstituted portions, and (c) fragments of the same. In certainembodiments, the tumor antigens are provided in recombinant form. Incertain embodiments, the tumor antigens include, for example, classI-restricted antigens recognized by CD8+ lymphocytes or classII-restricted antigens recognized by CD4+ lymphocytes.

In certain embodiments, the tumor antigens include, but are not limitedto, (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well asRAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1,GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12(which can be used, for example, to address melanoma, lung, head andneck, NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutatedantigens, for example, p53 (associated with various solid tumors, e.g.,colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g.,melanoma, pancreatic cancer and colorectal cancer), CDK4 (associatedwith, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspasc-8(associated with, e.g., head and neck cancer), CIA 0205 (associatedwith, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associatedwith, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkinslymphoma), BCR-abl (associated with, e.g., chronic myelogenousleukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT,(c) over-expressed antigens, for example, Galectin 4 (associated with,e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA(associated with, e.g., colorectal cancer), gastrin (associated with,e.g., pancreatic and gastric cancer), telomerase catalytic protein,MUC-1 (associated with, e.g., breast and ovarian cancer), G-250(associated with, e.g., renal cell carcinoma), p53 (associated with,e.g., breast, colon cancer), and carcinoembryonic antigen (associatedwith, e.g., breast cancer, lung cancer, and cancers of thegastrointestinal tract such as colorectal cancer), (d) shared antigens,for example, melanoma-melanocyte differentiation antigens such asMART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma), (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer, (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example), and (g)other tumor antigens, such as polypeptide- and saccharide-containingantigens including (i) glycoproteins such as sialyl Tn and sialyl Le^(x)(associated with, e.g., breast and colorectal cancer) as well as variousmucins; glycoproteins are coupled to a carrier protein (e.g., MUC-1 arecoupled to KLH); (ii) lipopolypeptides (e.g., MUC-1 linked to a lipidmoiety); (iii) polysaccharides (e.g., Globo H synthetic hexasaccharide),which are coupled to a carrier proteins (e.g., to KLH), (iv)gangliosides such as GM2, GM12, GD2, GD3 (associated with, e.g., brain,lung cancer, melanoma), which also are coupled to carrier proteins(e.g., KLH).

In certain embodiments, the tumor antigens include, but are not limitedto, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EpsteinBarr virus antigens, EBNA, human papillomavirus (HPV) antigens,including E6 and E7, hepatitis B and C virus antigens, human T-celllymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met,mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE,PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029,FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

Polynucleotide-containing antigens used in conjunction with theimmunogenic compositions provided herein include polynucleotides thatencode polypeptide cancer antigens such as those listed above. Incertain embodiments, the polynucleotide-containing antigens include, butare not limited to, DNA or RNA vector constructs, such as plasmidvectors (e.g., pCMV), which are capable of expressing polypeptide cancerantigens in vivo.

In certain embodiments, the tumor antigens are derived from mutated oraltered cellular components. After alteration, the cellular componentsno longer perform their regulatory functions, and hence the cell mayexperience uncontrolled growth. Representative examples of alteredcellular components include, but are not limited to ras, p53, Rb,altered protein encoded by the Wilms' tumor gene, ubiquitin, mucin,protein encoded by the DCC, APC, and MCC genes, as well as receptors orreceptor-like structures such as neu, thyroid hormone receptor, plateletderived growth factor (PDGF) receptor, insulin receptor, epidermalgrowth factor (EGF) receptor, and the colony stimulating factor (CSF)receptor.

Additionally, bacterial and viral antigens, are used in conjunction withthe immunogenic compositions provided herein for the treatment ofcancer. In certain embodiments, the, carrier proteins, such as CRM₁₉₇,tetanus toxoid, or Salmonella typhimurium antigen are used inconjunction/conjugation with compounds provided herein for treatment ofcancer. The cancer antigen combination therapies will show increasedefficacy and bioavailability as compared with existing therapies.

In certain embodiments, the immunogenic compositions containing at leastone compound of Formula (I) include capsular saccharides from at leasttwo of serogroups A, C, W135 and Y of Neisseria meningitides. In otherembodiments, such vaccines further comprise an antigen from one or moreof the following: (a) serogroup B N. meningitidis; (b) Haemophilusinfluenzae type B; and/or (c) Streptococcus pneumoniae.

In certain embodiments the immunogenic compositions containing at leastone compound of Formula (I) include serogroups C, W135 & Y of N.meningitides. In certain embodiments the immunogenic compositionscontaining at least one compound of Formula (I) include serogroups A, C,W135 & Y of N. meningitides. In certain embodiments the immunogeniccompositions containing at least one compound of Formula (I) includeserogroups B, C, W135 & Y of N. meningitides. In certain embodiments theimmunogenic compositions containing at least one compound of Formula (I)include serogroups A, B, C, W135 & Y of N. meningitides. In certainembodiments the immunogenic compositions containing at least onecompound of Formula (I) include H. influenzae type B and serogroups C,W135 & Y of N. meningitides. In certain embodiments the immunogeniccompositions containing at least one compound of Formula (I) include H.influenzae type B and serogroups A, C, W135 & Y of N. meningitides. Incertain embodiments the immunogenic compositions containing at least onecompound of Formula (I) include H. influenzae type B and serogroups B,C, W135 & Y of N. meningitides. In certain embodiments the immunogeniccompositions containing at least one compound of Formula (I) include H.influenzae type B and serogroups A, B, C, W135 & Y of N. meningitides.In certain embodiments the immunogenic compositions containing at leastone compound of Formula (I) include S. pneumoniae and serogroups C, W135& Y of N. meningitides. In certain embodiments the immunogeniccompositions containing at least one compound of Formula (I) include S.pneumoniae and serogroups A, C, W135 & Y of N. meningitides. In certainembodiments the immunogenic compositions containing at least onecompound of Formula (I) include S. pneumoniae and serogroups B, C, W135& Y of N. meningitides. In certain embodiments the immunogeniccompositions containing at least one compound of Formula (I) include S.pneumoniae and serogroups A, B, C, W135 & Y of N. meningitides. Incertain embodiments the immunogenic compositions containing at least onecompound of Formula (I) include H. influenzae type B, S. pneumoniae andserogroups C, W135 & Y of N. meningitides. In certain embodiments theimmunogenic compositions containing at least one compound of Formula (I)include H. influenzae type B, S. pneumoniae and serogroups A, C, W135 &Y of N. meningitides. In certain embodiments the immunogeniccompositions containing at least one compound of Formula (I) include H.influenzae type B, S. pneumoniae and serogroups B, C, W135 & Y of N.meningitides. In certain embodiments the immunogenic compositionscontaining at least one compound of Formula (I) include H. influenzaetype B, S. pneumoniae and serogroups A, B, C, W135 & Y of N.meningitidis.

5. Pharmaceutical Compositions and Administration

In another aspect, the invention provides a pharmaceutical compositioncomprising the homogeneous suspension of SMIP, and may further compriseone or more pharmaceutically acceptable carriers, diluents, orexcipients.

The pharmaceutical compositions provided herein may be administeredsingly or in combination with one or more additional therapeutic agents.The method of administration include, but are not limited to, oraladministration, rectal administration, parenteral, intravenousadministration, intravitreal administration, intramuscularadministration, inhalation, intranasal administration, topicaladministration, ophthalmic administration or otic administration.

The therapeutically effective amount of a SMIP will vary depending on,among others, the disease indicated, the severity of the disease, theage and relative health of the subject, the potency of the compoundadministered, the mode of administration and the treatment desired.

In other embodiments, the homogeneous suspension described herein, theimmunogenic composition described herein, or a pharmaceuticalcomposition thereof, is administered in combination with one or moreadditional therapeutic agents. The additional therapeutic agents mayinclude, but are not limited to antibiotics or antibacterial agents,antiemetics agents, antifungal agents, anti-inflammatory agents,antiviral agents, immunomodulatory agents, cytokines, antidepressants,hormones, alkylating agents, antimetabolites, antitumour antibiotics,antimitotic agents, topoisomerase inhibitors, cytostatic agents,anti-invasion agents, antiangiogenic agents, inhibitors of growth factorfunction inhibitors of viral replication, viral enzyme inhibitors,anticancer agents, α-interferons, β-interferons, ribavirin, hormones,and other toll-like receptor modulators, immunoglobulins (Igs), andantibodies modulating Ig function (such as anti-IgE (omalizumab)).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of infectious diseases including, but notlimited to, viral diseases such as genital warts, common warts, plantarwarts, respiratory syncytial virus (RSV), hepatitis B, hepatitis C,Dengue virus, herpes simplex virus (by way of example only, HSV-I,HSV-II, CMV, or VZV), molluscum contagiosum, vaccinia, variola,lentivirus, human immunodeficiency virus (HIV), human papilloma virus(HPV), cytomegalovirus (CMV), varicella zoster virus (VZV), rhinovirus,enterovirus, adenovirus, coronavirus (e.g., SARS), influenza,para-influenza, mumps virus, measles virus, papovavirus, hepadnavirus,flavivirus, retrovirus, arenavirus (by way of example only, LCM, Juninvirus, Machupo virus, Guanarito virus and Lassa Fever) and Filovirus (byway of example only, ebola virus or marbug virus).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of bacterial, fungal, and protozoal infectionsincluding, but not limited to, tuberculosis and mycobacterium avium,leprosy; pneumocystis carnii, cryptosporidiosis, histoplasmosis,toxoplasmosis, trypanosome infection, leishmaniasis, infections causedby bacteria of the genus Escherichia, Enterobacter, Salmonella,Staphylococcus, Klebsiella, Proteus, Pseudomonas, Streptococcus, andChlamydia, and fungal infections such as candidiasis, aspergillosis,histoplasmosis, cryptococcal meningitis.

In certain embodiments, the immunogenic compositions provided herein areused in the treatment of respiratory diseases and/or disorders,dermatological disorders, ocular diseases and/or disorders,genitourinary diseases and/or disorders including, allograft rejection,auto-immune and allergic, cancer, or damaged or ageing skin such asscarring and wrinkles.

In another aspect, the invention provides a method for generating animmune response in a subject in need thereof, such as a mammal,comprising administering an effective amount of an immunogeniccomposition as disclosed herein. The immune response is preferablyprotective and preferably involves antibodies and/or cell-mediatedimmunity. The method may raise a booster response.

In other aspects, the homogeneous suspensions and/or immunogeniccompositions provided herein are used to induce or potentiate an immuneresponse, for example, in the treatment of treatment of bacterial,fungal, and protozoal infections including, but not limited tomeneingococcemia, meningitis, meningococcal pheumonia, meningococcalpericarditis, meningococcal myocarditis, meningococcal pharyngitis,meningococcal conjunctivitis, meningococcal osteomyelitis, meningococcalendophthalmitis, meningococcal urethritis, Neisseria meningitidesinfection, tuberculosis and mycobacterium avium, leprosy; pneumocystiscarnii, cryptosporidiosis, histoplasmosis, toxoplasmosis, trypanosomeinfection, leishmaniasis, infections caused by bacteria of the genusEscherichia, Enterobacter, Salmonella, Staphylococcus, Klebsiella,Proteus, Pseudomonas, Streptococcus, and Chlamydia, and fungalinfections such as candidiasis, aspergillosis, histoplasmosis,cryptococcal meningitis.

In certain embodiments, the homogeneous suspensions and/or immunogeniccompositions provided herein are used to induce or potentiate an immuneresponse, for example, in the treatment of treatment of respiratorydiseases and/or disorders (e.g., RSV), meningococcal disease (e.g.,meneingococcemia, meningitis, meningococcal pheumonia, meningococcalpericarditis, meningococcal myocarditis, meningococcal pharyngitis,meningococcal conjunctivitis, meningococcal osteomyelitis, meningococcalendophthalmitis, meningococcal urethritis) dermatological disorders,ocular diseases and/or disorders, genitourinary diseases and/ordisorders including, allograft rejection, auto-immune and allergic,cancer, or damaged or ageing skin such as scarring and wrinkles.

In some preferred embodiments, the homogeneous suspensions and/orimmunogenic compositions provided herein are used to induce orpotentiate an immune response, e.g., a protective immune response,against RSV, Ebola, or N. meningitides.

In some embodiments, the invention provides a method for generating orpotentiating an immune response, comprising administering an effectiveamount of an immunogenic composition or a homogeneous suspension, asdescribed herein, to a subject in need thereof. The immune response canbe a naturally occurring immune response, or an induced immune response,for example, induced by immunization.

When a homogeneous suspension of the Benzonaphthyridine compound offormula I and II is administered to potentiate an induced immuneresponse, it is preferred that it is administered at substantially thesame time as the agent that induces the immune response. For example, aneffective amount of a homogeneous suspension can be administeredconcurrently with an immunogenic composition or vaccine, or administeredwithin a period of about 1 day before or after the immunogeniccomposition or vaccine is administered.

In certain embodiments, if an aluminum-containing adjuvant is used, itmay be desirable to package the alum-adsorbed antigen and thehomogeneous suspension of the Benzonaphthyridine compound of formula Iand II separately. The two components may be combined, e.g., withinabout 72 hours prior to administration, so that the desorption of theantigen from the aluminum-containing adjuvant is reduced. For example,the alum-adsorbed antigen and the homogeneous suspension can be combinedat a patient's bedside. Preferably, the alum-adsorbed antigen and thehomogeneous suspension are combined within about 72 hours, about 48hours, about 24 hours, about 12 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4hours, about 3 hours, about 2 hours, about 1 hour, about 45 minutes,about 30 minutes, about 15 minutes, about 10 minutes, or about 5 minutesprior to administration.

In certain embodiments, the immunogenic compositions disclosed hereinmay be used as a medicament, e.g., for use in raising or enhancing animmune response in a subject in need thereof, such as a mammal.

In certain embodiments, the immunogenic compositions disclosed hereinmay be used in the manufacture of a medicament for raising an immuneresponse in a subject in need thereof, such as a mammal. The inventionalso provides a delivery device pre-filled with an immunogeniccomposition disclosed herein.

The mammal is preferably a human, but may be, e.g., a cow, a pig, achicken, a cat or a dog, as the pathogens covered herein may beproblematic across a wide range of species. Where the vaccine is forprophylactic use, the human is preferably a child (e.g., a toddler orinfant) or a teenager; where the vaccine is for therapeutic use, thehuman is preferably a teenager or an adult. A vaccine intended forchildren may also be administered to adults, e.g., to assess safety,dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring pathogen infection after administration of the immunogeniccompositions disclosed herein. One way of checking efficacy ofprophylactic treatment involves monitoring immune responses,systemically (such as monitoring the level of IgG1 and IgG2a production)and/or mucosally (such as monitoring the level of IgA production),against the antigens included in or administered in conjunction with theimmunogenic compositions disclosed herein after administration of theimmunogenic composition (and the antigen if administered separately).Typically, antigen-specific serum antibody responses are determinedpost-immunisation but pre-challenge whereas antigen-specific mucosalantibody responses are determined post-immunisation and post-challenge.

Another way of assessing the immunogenicity of the immunogeniccompositions disclosed herein where the antigen is a protein is toexpress the proteins recombinantly for screening patient sera or mucosalsecretions by immunoblot and/or microarrays. A positive reaction betweenthe protein and the patient sample indicates that the patient hasmounted an immune response to the protein in question. This method mayalso be used to identify immunodominant antigens and/or epitopes withinprotein antigens.

The efficacy of the immunogenic compositions can also be determined invivo by challenging appropriate animal models of the pathogen ofinterest infection.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

The immunogenic compositions disclosed herein that include one or moreantigens or are used in conjunction with one or more antigens may beused to treat both children and adults. Thus a human subject may be lessthan 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or atleast 55 years old. Preferred subjects for receiving such immunogeniccompositions are the elderly (e.g., >50 years old, >60 years old, andpreferably >65 years), the young (e.g., <5 years old), hospitalisedpatients, healthcare workers, armed service and military personnel,pregnant women, the chronically ill, or immunodeficient patients. Theimmunogenic compositions are not suitable solely for these groups,however, and may be used more generally in a population.

The immunogenic compositions disclosed herein that include one or moreantigens or are used in conjunction with one or more antigens may beadministered to patients at substantially the same time as (e.g., duringthe same medical consultation or visit to a healthcare professional orvaccination centre) other vaccines, e.g., at substantially the same timeas a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine,a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzaetype b vaccine, an inactivated poliovirus vaccine, a hepatitis B virusvaccine, a meningococcal conjugate vaccine (such as a tetravalent A CW135 Y vaccine), a respiratory syncytial virus vaccine, etc.

6. Kits

The invention also provides kits comprising a homogeneous suspension anda composition comprising an antigen in separate containers. For example,the kit can contain a first container comprising a homogeneoussuspension, and a second container comprising a composition thatcomprises an antigen. The composition that comprises an antigen can bein liquid form or can be in solid form (e.g., lyophilized), as canindividual antigens. Suitable containers for the compositions include,for example, bottles, vials, syringes, and test tubes. Containers can beformed from a variety of materials, including glass or plastic. Acontainer may have a sterile access port (for example, the container maybe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle).

The kit can further comprise a third container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to the end-user, including other pharmaceuticallyacceptable formulating solutions such as buffers, diluents, filters,needles, and syringes or other delivery device. The kit may furtherinclude a fourth container comprising an adjuvant (such as an aluminumcontaining adjuvant or MF59).

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity or for treatinginfections. The package insert can be an unapproved draft package insertor can be a package insert approved by the Food and Drug Administration(FDA) or other regulatory body.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example (I) Preparation of Selected Benzo[f][1,7]naphthyridin-5-amineanalogs

The following examples illustrate methods for preparing certaincompounds useful in the compositions and methods of the invention. Theskilled person would be able to make a wide range of other compounds foruse in the instant methods based on these examples.

Example 1 Benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-bromopicolino-nitrile (1.0 eq.) in toluene (0.44 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and 2N aqueouspotassium carbonate solution (2.0 eq.). The reaction was heated to 100°C. and stirred overnight. After cooling to ambient temperature, thereaction content was diluted with 2% methanol in dichloromethane andwater. The two phases were separated, and the aqueous layer wasextracted twice with 2% methanol in dichloromethane. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vacuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-80% ethyl acetatein hexane to give a white solid. ¹H NMR (acetone d-6): δ 9.04 (d, 1H),8.91 (d, 1H), 8.45 (d, 1H), 7.86 (dd, 1H), 7.53-7.62 (m, 2H), 7.35 (t,1H), 6.65 (br, 2H). LRMS [M+H]=196.1

Example 3 9-chlorobenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-4-chlorophenylcarbamate

To a solution of 2-bromo-4-chloroaniline (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified y byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give the product as light yellow oil.

Step 2: tert-butyl4-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-carbamate

Tert-butyl 2-bromo-4-chlorophenylcarbamate (from step 1) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude mixture was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ethyl acetate in hexane to givetert-butyl4-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-carbamate.

Step 3: 9-chlorobenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl4-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-carbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-50% ethylacetate in hexane and then re-purified using 0-5% methanol indichloromethane to give a solid. ¹H NMR (acetone d-6): δ 9.08 (d, 1H),8.96 (d, 1H), 8.45 (s, 1H), 7.86-7.89 (dd, 1H), 7.60 (d, 1H), 7.54 (d,1H), 6.78 (br, 2H). LRMS [M+H]=230.1

Example 4 8-chlorobenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-5-chlorophenylcarbamate

To a solution of 2-bromo-5-chloroaniline (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give the product as light yellow oil.

Step 2: tert-butyl5-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-carbamate

Tert-butyl 2-bromo-5-chlorophenylcarbamate (from step 1) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude mixture was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-5% ethyl acetate in hexane to givetert-butyl5-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-carbamate.

Step 3: 8-chlorobenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-carbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% methanolin dichloromethane to give a semipure solid, which was then stirred inhot 10% ethyl acetate in hexane, filtered, and dried to give a puresolid. ¹H NMR (acetone d-6): δ 9.03 (d, 1H), 8.93 (d, 1H), 8.46 (d, 1H),7.85-7.88 (dd, 1H), 7.57 (s, 1H), 7.32 (d, 1H), 6.94 (br, 2H). LRMS[M+H]=230.1

Example 5 8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-5-methylphenylcarbamate

To a solution of 2-bromo-5-methylaniline (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give product as light yellow oil.

Step 2: tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-bromo-5-methylphenylcarbamate (from step 1) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-8% ether in hexane to give tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate.

Step 3: 8-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-40% ethylacetate in toluene to give a pure solid. ¹H NMR (acetone d-6): δ 8.98(d, 1H), 8.87 (d, 1H), 8.32 (d, 1H), 7.79-7.82 (dd, 1H), 7.42 (s, 1H),7.18 (d, 1H), 6.6 (br, 2H), 2.45 (s, 3H). LRMS [M+H]=210.1

Example 6 9-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-4-methylphenylcarbamate

To a solution of 2-bromo-4-methylaniline (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give product as light yellow oil.

Step 2: tert-butyl4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-bromo-4-methylphenylcarbamate (from step 1) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-8% ether in hexane to give tert-butyl4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate.

Step 3: 9-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% methanolin dichloromethane to give a semipure solid, which was then swirled inhot ethyl acetate, filtered, and dried to give a pure solid. ¹H NMR(acetone d-6): δ 9.02 (d, 1H), 8.89 (d, 1H), 8.25 (s, 1H), 7.80-7.84(dd, 1H), 7.52 (d, 1H), 7.40 (d, 1H), 6.5 (br, 2H), 2.48 (s, 3H). LRMS[M+H]=210.2

Example 7 10-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-3-methylphenylcarbamate

To a solution of 2-bromo-3-methylaniline (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give product as light yellow oil.

Step 2: tert-butyl3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-bromo-3-methylphenylcarbamate (from step 1) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ether in hexane to tert-butyl3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate.

Step 3: 10-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-40% ethylacetate in toluene to give a semipure solid, which was then swirled inhot 10% ethyl acetate in hexane, filtered, and dried to give a puresolid. ¹H NMR (acetone d-6): δ 9.22 (d, 1H), 8.90 (d, 1H), 7.82-7.85(dd, 1H), 7.54 (d, 1H), 7.45 (t, 1H), 7.19 (d, 1H), 6.6 (br, 2H), 2.98(s, 3H). LRMS [M+H]=210.2.

Example 8 Ethyl 5-aminobenzo[f][1,7]naphthyridine-9-carboxylate

Step 1: ethyl 3-bromo-4-(tert-butoxycarbonylamino)benzoate

To a solution of 4-amino-3-bromobenzoate (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give product as light yellow oil.

Step 2: Ethyl4-(tert-butoxycarbonylamino)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate

Ethyl 3-bromo-4-(tert-butoxycarbonylamino)benzoate (from step 1) (1.0eq.), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5eq.), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%),and sodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ether in hexane to give ethyl4-(tert-butoxycarbonylamino)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate.

Step 3: ethyl 5-aminobenzo[f][1,7]naphthyridine-9-carboxylate

A solution of ethyl4-(tert-butoxycarbonylamino)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) intoluene/ethanol (10:1, 0.23 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and anhydrous potassiumcarbonate (2.0 eq.). The reaction was heated to 100° C. and stirredovernight. After cooling to ambient temperature, the reaction contentwas diluted with 2% methanol in dichloromethane and water. The twophases were separated, and the aqueous layer was extracted twice with 2%methanol in dichloromethane. The combined organic layers were washedwith brine, dried over anhydrous MgSO₄, and concentrated en vacuo. Thecrude material was purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-40% ethyl acetate in toluene to give a semipuresolid, which was then swirled in hot 10% ethyl acetate in hexane,filtered, and dried to give a pure solid. ¹H NMR (acetone d-6): δ 9.11(d, 1H), 9.05 (s, 1H), 8.95 (d, 1H), 8.14 (d, 1H), 7.89-7.92 (dd, 1H),7.63 (d, 1H), 4.38 (q, 2H), 1.40 (t, 3H). LRMS [M+H]=268.2.

Example 9 5-aminobenzo[f][1,7]naphthyridine-9-carboxylic acid

Ethyl 5-aminobenzo[f][1,7]naphthyridine-9-carboxylate (Example 8) (1.0eq.) was mixed with 1N NaOH (2.0 eq.) in ethanol (0.12 M). The reactionwas heated to 80° C. and stirred for 36 hours. The solvent was removeden vacuo. The residue was suspended in water, and the pH was adjusted toneutral using 5% citric acid aqueous solution. The suspension wascentrifuged (2500 rpm, 5 min), and the supernatant was removed. Theresulting solids was re-suspended in water by vortexing, centrifuged(2500 rpm, 5 min), and the supernatant was removed. The re-suspension,centrifugation, and removal of supernatant steps were repeated with hotmethanol, hot ethyl acetate, and ether to give a pure solid. ¹H NMR(DMSO): δ 12.86 (s, 1H), 9.15 (d, 1H), 9.00 (s, 1H), 8.97 (d, 1H), 8.07(d, 1H), 7.88-7.91 (dd, 1H), 7.56-7.59 (m, 3H). LRMS [M+H]=240.1

Example 10 8-methoxybenzo[f][1,7]naphthyridin-5-amine

Step 1: 2-bromo-5-methoxyaniline

A solution of 1-bromo-4-methoxy-2-nitrobenzene (1.0 eq.), iron powder(3.0 eq.), and concentrated HCl (1.04 eq.) were mixed together inethanol (0.64 M) and heated to reflux. The reaction was stirred for 24hours, and the solvent was evaporated. The resulting residue was dilutedwith ethyl acetate and saturated aqueous ammonium chloride solution. Theaqueous layer was extracted three times with ethyl acetate, and thecombined organic layers were washed with water, brine, dried overanhydrous MgSO₄, and concentrated en vacuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-15% ethyl acetate in hexane to give the product as oil.

Step 2: tert-butyl 2-bromo-5-methoxyphenylcarbamate

To a solution of 2-bromo-5-methoxyaniline (1.0 eq.) (from step 1) intetrahydrofuran (0.2 M) at 0° C. under N₂ atmosphere was added dropwise1M NaHMDS (2.5 eq.). The reaction was stirred for 15 minutes at 0° C.,and a solution of di-tert-butyl dicarbonate in tetrahydrofuran wasadded. The reaction was warmed to room temperature overnight. Thesolvent was evaporated, and the resulting residue was quenched with 0.1NHCl aqueous solution. The aqueous suspension was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vacuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-5% ethyl acetate in hexane to give product as light yellow oil.

Step 3: tert-butyl5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-bromo-5-methoxyphenylcarbamate (from step 2) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-15% ether in hexane to give tert-butyl5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate.

Step 4: 8-methoxybenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from step 3) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) intoluene/ethanol (10:1, 0.23 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and anhydrous potassiumcarbonate (2.0 eq.). The reaction was heated to 100° C. and stirredovernight. After cooling to ambient temperature, the reaction contentwas diluted with 2% methanol in dichloromethane and water. The twophases were separated, and the aqueous layer was extracted twice with 2%methanol in dichloromethane. The combined organic layers were washedwith brine, dried over anhydrous MgSO₄, and concentrated en vacuo. Thecrude product was purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-5% methanol in dichloromethane to give a semipuresolid, which was then recrystallized in ethyl acetate, filtered, anddried to give a pure solid. ¹H NMR (acetone d-6): δ 8.91 (d, 1H), 8.82(d, 1H), 8.33 (d, 1H), 7.76-7.79 (dd, 1H), 7.07 (s, 1H), 6.96 (d, 1H),6.6 (br, 2H), 3.90 (s, 3H).

LRMS [M+H]=226.1

Example 11 7-fluorobenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-fluorophenylcarbamate

To a solution of 2-fluoroaniline (1.0 eq.) in tetrahydrofuran (0.2 M) at0° C. under N2 atmosphere was added dropwise 1M NaHMDS (2.5 eq.). Thereaction was stirred for 15 minutes at 0° C., and a solution ofdi-tert-butyl dicarbonate in tetrahydrofuran was added. The reaction waswarmed to room temperature overnight. The solvent was evaporated, andthe resulting residue was quenched with 0.1N HCl aqueous solution. Theaqueous suspension was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vacuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-5% ethyl acetatein hexane to give product as light yellow oil.

Step 2: 2-(tert-butoxycarbonylamino)-3-fluorophenylboronic acid

To a solution of tert-butyl 2-fluorophenylcarbamate (from step 1) (1.0eq.) in tetrahydrofuran (0.25 M) at −78° C. under N₂ atmosphere wasadded dropwise 1.7 M tert-butyllithium (2.4 eq.). The reaction waswarmed to −40° C. slowly over 2 hours, and neat trimethyl borate (3.8eq.) was added. The reaction was warmed to room temperature over 30minutes. An aqueous solution of 1N NaOH was slowly added to the reactionand stirred for 15 minutes. The mixture was poured into ethyl acetateand acidified with 3N HCl to dissolve the solids. The aqueous layer wasextracted twice with ethyl acetate, and the combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envacuo. The resulting solids were stirred in 1:1 ether/hexane, filtered,and dried. The solids were carried onto the next step without furtherpurification.

Step 3: 7-fluorobenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)-3-fluorophenylboronic acid(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. After workup, the crude product wassuspended in hot toluene, centrifuged (2500 rpm, 5 min), and thesupernatant was removed. The suspension, centrifugation, and removal ofsupernatant steps were repeated with hot ethyl acetate, ether, andhexane to give a pure solid. ¹H NMR (acetone d-6): δ 9.04 (d, 1H), 8.96(d, 1H), 8.27 (d, 1H), 7.86-7.90 (dd, 1H), 7.28-7.34 (m, 2H), 6.9 (br,2H). LRMS [M+H]=214.1

Example 12 8-(methylsulfonyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 2-bromo-5-(methylsulfonyl)aniline

A solution of 1-bromo-4-(methylsulfonyl)-2-nitrobenzene (1.0 eq.), ironpowder (3.0 eq.), and concentrated HCl (1.04 eq.) were mixed together inethanol (0.64 M) and heated to reflux. The reaction was stirred for 24hours, and the solvent was evaporated. The resulting residue was dilutedwith ethyl acetate and saturated aqueous ammonium chloride solution. Theaqueous layer was extracted three times with ethyl acetate, and thecombined organic layers were washed with water, brine, dried overanhydrous MgSO₄, and concentrated en vacuo. The crude material waspurified by triturating in 1:1 hexane/ether to give a light yellowsolid.

Step 2: tert-butyl 2-bromo-5-(methylsulfonyl)phenylcarbamate

To a solution of 2-bromo-5-(methylsulfonyl)aniline (from step 1) (1.0eq.) in tetrahydrofuran (0.2 M) at 0° C. under N₂ atmosphere was addeddropwise 1M NaHMDS (2.5 eq.). The reaction was stirred for 15 minutes at0° C., and a solution of di-tert-butyl dicarbonate in tetrahydrofuranwas added. The reaction was warmed to room temperature overnight. Thesolvent was evaporated, and the resulting residue was quenched with 0.1NHCl aqueous solution. The aqueous suspension was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vacuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-5% ethyl acetate in hexane to give tert-butyl2-bromo-5-(methylsulfonyl)phenylcarbamate.

Step 3: tert-butyl5-(methylsulfonyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-bromo-5-(methylsulfonyl)phenylcarbamate (from step 2) (1.0eq.), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5eq.), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%),and sodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-50% ethyl acetate in hexane to give asolid which was then triturated in 10% ether/hexane to give tert-butyl5-(methylsulfonyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamateas a white solid.

Step 4: 8-(methylsulfonyl)benzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-(methylsulfonyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from step 3) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.24 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (4.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% methanolin dichloromethane to give a solid which was then triturated in 1:1hexane/ethyl acetate to give8-(methylsulfonyl)benzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetoned-6): δ 9.16 (d, 1H), 9.03 (d, 1H), 8.71 (d, 1H), 8.11 (s, 1H),7.93-7.96 (dd, 1H), 7.81 (d, 1H), 7.0 (br, 2H), 3.19 (s, 3H). LRMS[M+H]=274.1

Example 13 8-(trifluoromethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-5-(trifluoromethyl)phenylcarbamate

To a solution of 2-bromo-5-(trifluoromethyl)aniline (1.0 eq.) intetrahydrofuran (0.2 M) at 0° C. under N₂ atmosphere was added dropwise1M NaHMDS (2.5 eq.). The reaction was stirred for 15 minutes at 0° C.,and a solution of di-tert-butyl dicarbonate in tetrahydrofuran wasadded. The reaction was warmed to room temperature overnight. Thesolvent was evaporated, and the resulting residue was quenched with 0.1NHCl aqueous solution. The aqueous suspension was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vacuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-5% ethyl acetate in hexane to give product as light yellow oil.

Step 2: tert-butyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenylcarbamate

Tert-butyl 2-bromo-5-(trifluoromethyl)phenylcarbamate (from step 1) (1.0eq.), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5eq.), dichloro[1′,1′-bis(diphenylphosphino)ferrocene]palladium (II)(5%), and sodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) underN₂ atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ether in hexane to give an impureproduct which was carried onto the next step without furtherpurification.

Step 3: 8-(trifluoromethyl)benzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenylcarbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.24 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (4.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-40% ethylacetate in toluene to give a solid which was then triturated in 10%ethyl acetate in hexane to give8-(trifluoromethyl)benzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetoned-6): δ 9.13 (d, 1H), 9.00 (d, 1H), 8.67 (d, 1H), 7.91-7.94 (dd, 1H),7.86 (s, 1H), 7.58 (d, 1H), 6.9 (br, 2H). LRMS [M+H]=264.1

Example 14 8-fluorobenzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-bromo-5-fluorophenylcarbamate

To a solution of 2-bromo-5-fluoroaniline (1.0 eq.) in tetrahydrofuran(0.2 M) at 0° C. under N₂ atmosphere was added dropwise 1M NaHMDS (2.5eq.). The reaction was stirred for 15 minutes at 0° C., and a solutionof di-tert-butyl dicarbonate in tetrahydrofuran was added. The reactionwas warmed to room temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in hexane to give the product as light yellow oil.

Step 2: tert-butyl5-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-bromo-5-fluorophenylcarbamate (from step 1) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.5 eq.),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) (5%), andsodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) under N₂atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-5% ether in hexane to give the productas a yellow solid.

Step 3: 8-fluorobenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from step 2) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.) in toluene(0.24 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (4.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-40% ethylacetate in toluene to give a solid which was then triturated in 10%ethyl acetate in hexane to give8-fluorobenzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetone d-6): δ 9.00(d, 1H), 8.90 (d, 1H), 8.46-8.50 (dd, 1H), 7.83-7.87 (dd, 1H), 7.26 (d,1H), 7.15 (t, 1H), 6.9 (br, 2H). LRMS [M+H]=214.1

Example 16 3-methoxybenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-bromo-6-methoxypicolinonitrile

A solution of 3-bromo-6-oxo-1,6-dihydropyridine-2-carbonitrile (fromExample 15/Step 2) (1.0 eq.), silver carbonate (1.3 eq.), andiodomethane (1.2 eq.) in toluene (0.2 M) was stirred in the dark at roomtemperature overnight. The solvent was concentrated en vacuo, and theresulting residue was purified by a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to give 3-bromo-6-methoxypicolinonitrile.

Step 2: 3-methoxybenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-bromo-6-methoxypicolinonitrile (from step 1) (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-50% ethylacetate in hexane to give 3-methoxybenzo[f][1,7]naphthyridin-5-amine asa yellow solid. ¹H NMR (acetone d-6): δ 8.91 (d, 1H), 8.34 (d, 1H), 7.63(d, 1H), 7.51-7.53 (dd, 1H), 7.27-7.33 (m, 2H), 6.65 (br, 2H), 4.11 (s,3H). LRMS [M+H]=226.1

Example 17 3-butoxybenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-bromo-6-butoxypicolinonitrile

A solution of 3-bromo-6-oxo-1,6-dihydropyridine-2-carbonitrile (fromExample 15/Step 2) (1.0 eq.), potassium carbonate (1.3 eq.), and1-iodobutane (1.2 eq.) in acetone (0.3 M) was stirred at 70° C.overnight. The solvent was concentrated en vacuo, and the resultingresidue was taken up in water and ethyl acetate. The aqueous layer wasextracted with ethyl acetate three times. The combined organic layerswere washed with brine, dried over anhydrous MgSO₄, and concentrated envacuo. The crude product was purified by a COMBIFLASH® system (ISCO)using 0-30% ethyl acetate in hexane to give a colorless solid.

Step 2: 3-butoxybenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-bromo-6-butoxypicolinonitrile (from step 1) (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5% ethylacetate in methanol to give 3-butoxybenzo[f][1,7]naphthyridin-5-amine asa white solid. ¹H NMR (acetone d-6): δ 8.91 (d, 1H), 8.34 (d, 1H), 7.61(d, 1H), 7.48-7.52 (dd, 1H), 7.27-7.33 (m, 2H), 6.51 (br, 2H), 6.55 (t,2H), 1.81-1.88 (m, 2H), 1.50-1.59 (m, 2H), 1.00 (t, 3H). LRMS[M+H]=268.1

Example 18 3-(benzyloxy)benzo[f][1,7]naphthyridin-5-amine

Step 1: 6-(benzyloxy)-3-bromopicolinonitrile

A solution of 3-bromo-6-oxo-1,6-dihydropyridine-2-carbonitrile (fromExample 15/Step 2) (1.0 eq.), silver carbonate (1.3 eq.), and benzylbromide (1.2 eq.) in toluene (0.16 M) was stirred in the dark at 50° C.overnight. The solvent was concentrated en vacuo, and the resultingresidue was purified by a COMBIFLASH® system (ISCO) using 0-20% ethylacetate in hexane to give 6-(benzyloxy)-3-bromopicolinonitrile.

Step 2: 3-(benzyloxy)benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 6-(benzyloxy)-3-bromopicolinonitrile (from step 1) (1.0 eq.) intoluene (0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium(5 mol %) and 2N aqueous potassium carbonate solution (2.0 eq.). Thereaction was heated to 100° C. and stirred overnight. After cooling toambient temperature, the reaction content was diluted with 2% methanolin dichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-50% ethylacetate in hexane to give 3-(benzyloxy)benzo[f][1,7]naphthyridin-5-amineas a yellow solid. ¹H NMR (acetone d-6): δ 8.95 (d, 1H), 8.35 (d, 1H),7.58-7.63 (m, 2H), 7.49-7.53 (dd, 1H), 7.30-7.44 (m, 5H), 6.61 (br, 2H),5.64 (s, 2H). LRMS [M+H]=302.1

Example 19 3-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 5-bromo-2-methylpyridine 1-oxide

To a solution of 5-bromo-2-methylpyridine (1.0 eq.) in chloroform (0.38M) was added 77% meta-chloroperbenzoic acid (mCPBA) (4.0 eq.) and heatedat 60° C. for 20 hours. After cooling to room temperature, Ca(OH)₂ (5.3eq.) was added, and the resulting precipitate was stirred for 30minutes. The precipitate was filtered and washed with 3:1CHCl₃/methanol. The filtrate was concentrated en vacuo to give a solid,which was stirred in 30% ethyl acetate in hexane and filtered to givethe desired N-oxide. The filtrate was concentrated en vacuo, and theresidue was purified by a COMBIFLASH® system (ISCO) using 0-100% ethylacetate in hexane to give more of the desired N-oxide. The two batcheswere combined and carried onto the next step.

Step 2: 3-bromo-6-methylpicolinonitrile

To a solution of 5-bromo-2-methylpyridine 1-oxide (from step 1) (1.0eq.) in acetonitrile (0.2 M) was added trimethylsilyl cyanide (TMSCN)(4.0 eq.) and triethylamine (3.0 eq.). The reaction was heated at 100°C. overnight. After cooling to room temperature, the solvent wasconcentrated en vacuo, and the residue was purified by a COMBIFLASH®system (ISCO) using 0-50% ethyl acetate in hexane to give3-bromo-6-methylpicolinonitrile.

Step 3: 3-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-bromo-6-methylpicolinonitrile (from step 2) (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-70% ethylacetate in hexane to give 3-methylbenzo[f][1,7]naphthyridin-5-amine as ayellow solid. ¹H NMR (methanol d-4): δ 8.85 (d, 1H), 8.38 (d, 1H), 7.72(d, 1H), 7.53-7.61 (m, 2H), 7.34-7.38 (dd, 1H), 2.76 (s, 3H). LRMS[M+H]=210.1

Example 20 3-chlorobenzo[f][1,7]naphthyridin-5-amine

Step 1: 5-bromo-2-chloropyridine 1-oxide

To a solution of 5-bromo-2-chloropyridine (1.0 eq.) in chloroform (0.38M) was added 77% meta-chloroperbenzoic acid (mCPBA) (4.0 eq.) and heatedat 60° C. for 20 hours. After cooling to room temperature, Ca(OH)₂ (5.3eq.) was added, and the resulting precipitate was stirred for 30minutes. The precipitate was filtered and washed with 3:1CHCl₃/methanol. The filtrate was concentrated en vacuo to give a solid,which was stirred in 30% ethyl acetate in hexane and filtered to givethe desired N-oxide. The filtrate was concentrated en vacuo, and theresidue was purified by a COMBIFLASH® system (ISCO) using 0-100% ethylacetate in hexane to give more of the desired N-oxide. The two batcheswere combined and carried onto the next step.

Step 2: 3-bromo-6-chloropicolinonitrile

To a solution of 5-bromo-2-chloropyridine 1-oxide (from step 1) (1.0eq.) in acetonitrile (0.2 M) was added trimethylsilyl cyanide (TMSCN)(4.0 eq.) and triethylamine (3.0 eq.). The reaction was heated at 100°C. overnight. After cooling to room temperature, the solvent wasconcentrated en vacuo, and the residue was purified by a COMBIFLASH®system (ISCO) using 0-40% ethyl acetate in hexane to give3-bromo-6-chloropicolinonitrile.

Step 3: 3-chlorobenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-bromo-6-chloropicolinonitrile (from step 2) (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-50% ethylacetate in hexane to give a solid, which was then triturated in 10%ethyl acetate in hexane to give3-chlorobenzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetone d-6): δ 9.10(d, 1H), 8.45 (d, 1H), 7.89 (d, 1H), 7.58-7.65 (m, 2H), 7.35-7.39 (dd,1H), 6.67 (br, 2H). LRMS [M+H]=230.1

Example 21

N³,N³-dimethylbenzo[f][1,7]naphthyridine-3,5-diamine

A solution of 3-chlorobenzo[f][1,7]naphthyridin-5-amine (Example 20)(1.0 eq.) was dissolved in 40% aqueous dimethylamine (0.26 M) and heatedin a microwave reactor at 100° C. for 30 minutes. The reaction mixturewas concentrated en vacuo, and the residue was purified by a COMBIFLASH®system (ISCO) using 0-90% ethyl acetate in hexane to giveN³,N³-dimethylbenzo[f][1,7]naphthyridine-3,5-diamine. ¹H NMR (methanold-4): δ 8.63 (d, 1H), 8.20 (d, 1H), 7.55 (d, 1H), 7.41-7.45 (dd, 1H),7.29-7.33 (dd, 1H), 7.27 (d, 1H), 3.26 (s, 6H). LRMS [M+H]=239.1

Example 22

N³-butylbenzo[f][1,7]naphthyridine-3,5-diamine

A solution of 3-chlorobenzo[f][1,7]naphthyridin-5-amine (Example 20)(1.0 eq.) was dissolved in n-butylamine (0.1 M) and heated at 110° C.overnight. The reaction mixture was concentrated en vacuo, and theresidue was purified by a COMBIFLASH® system (ISCO) using 0-90% ethylacetate in hexane to giveN³-butylbenzo[f][1,7]naphthyridine-3,5-diamine. ¹H NMR (methanol d-4): δ8.42 (d, 1H), 8.13 (d, 1H), 7.53 (d, 1H), 7.38-7.42 (dd, 1H), 7.25-7.29(dd, 1H), 6.96 (d, 1H), 3.48 (t, 2H), 1.63-1.71 (m, 2H), 1.43-1.52 (m,2H), 0.99 (t, 3H). LRMS [M+H]=267.2

Example 23 3-vinylbenzo[f][1,7]naphthyridin-5-amine

A solution of 3-chlorobenzo[f][1,7]naphthyridin-5-amine (Example 20)(1.0 eq.), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (1.2 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueouspotassium carbonate solution (2.0 eq.) in toluene/ethanol (4:1, 0.1 M)was heated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with ethyl acetate andwater. The two phases were separated, and the aqueous layer wasextracted with ethyl acetate three times. The combined organic layerswere washed with brine, dried over anhydrous MgSO₄, and concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-50% ethyl acetate in hexane to give asolid, which was then triturated in 10% ethyl acetate in hexane to give3-vinylbenzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetone d-6): δ 8.99(d, 1H), 8.42 (d, 1H), 8.01 (d, 1H), 7.53-7.62 (m, 2H), 7.30-7.35 (dd,1H), 7.03-7.10 (dd, 1H), 6.77 (br, 2H), 6.56 (d, 1H), 5.66 (d, 1H). LRMS[M+H]=222.1

Example 24 3-ethylbenzo[f][1,7]naphthyridin-5-amine

To a solution of 3-vinylbenzo[f][1,7]naphthyridin-5-amine (Example 23)in ethyl acetate/ethanol (1:1, 0.07 M) was added 10% wt palladium oncarbon (0.2 eq.). Hydrogen gas was introduced via a balloon, and thereaction was stirred overnight. The mixture was filtered through a padof celite, washing with dichloromethane. The filtrate was concentrateden vacuo giving 3-ethylbenzo[f][1,7]naphthyridin-5-amine as a whitesolid. ¹H NMR (acetone d-6): δ 8.93 (d, 1H), 8.41 (d, 1H), 7.76 (d, 1H),7.61 (d, 1H), 7.51-7.55 (dd, 1H), 7.30-7.34 (dd, 1H), 6.55 (br, 2H),6.03 (q, 2H), 1.41 (t, 3H). LRMS [M+H]=224.1

Example 25 3-fluorobenzo[f][1,7]naphthyridin-5-amine

A solution of 3-chlorobenzo[f][1,7]naphthyridin-5-amine (Example 20)(1.0 eq.), potassium fluoride (3.0 eq.), and 18-crown-6 (0.2 eq.) inN-methylpyrrolidone (NMP) (0.4 M) was heated in a microwave reactor at210° C. for 80 minutes. After cooling to room temperature, the crudereaction mixture was purified by HPLC using 10-50% acetonitrile in waterto give 3-fluorobenzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetone d-6):δ 11.40 (br, 2H), 9.38-9.42 (dd, 1H), 8.60 (d, 1H), 7.89-7.92 (dd, 1H),7.81-7.83 (m, 2H), 7.59-7.66 (m, 1H).

LRMS [M+H]=214.1

Example 26 2-(trifluoromethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-(trifluoromethyl)picolinaldehyde oxime

A solution of 3-chloro-5-(trifluoromethyl)picolinaldehyde (1.0 eq.),hydroxylamine hydrochloride (5.0 eq.), and pyridine (4.0 eq.) in ethanolwas heated to 95° C. and stirred for 1 hour. The reaction was cooled toroom temperature and diluted with ethyl acetate and water. The organiclayer was washed with brine, water, dried over anhydrous MgSO₄, andconcentrated en vacuo to give a solid that was carried onto the nextstep without further purification.

Step 2: 3-chloro-5-(trifluoromethyl)picolinonitrile

A solution of 3-chloro-5-(trifluoromethyl)picolinaldehyde oxime (1.0eq.) and Burgess reagent (1.5 eq.) in tetrahydrofuran (0.5 M) was heatedto 65° C. and stirred for 1 hour. The reaction was cooled to roomtemperature and diluted with ethyl acetate and water. The organic layerwas washed with water, brine, dried over anhydrous MgSO₄, andconcentrated en vacuo to give a solid that was carried onto the nextstep without further purification.

Step 3: 2-(trifluoromethyl)benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-chloro-5-(trifluoromethyl)picolinonitrile (from step 2) (1.0 eq.)in toluene (0.44 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and 2N aqueouspotassium carbonate solution (2.0 eq.). The reaction was heated to 100°C. and stirred overnight. After cooling to ambient temperature, thereaction content was diluted with 2% methanol in dichloromethane andwater. The two phases were separated, and the aqueous layer wasextracted twice with 2% methanol in dichloromethane. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vacuo. The crude product was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-50% ethyl acetatein hexane to give 2-(trifluoromethyl)benzo[f][1,7]naphthyridin-5-amine.¹H NMR (acetone d-6): δ 9.44 (s, 1H), 9.20 (s, 1H), 8.65-8.63 (d, 1H),7.70-7.61 (m, 2H), 7.44-7.36 (m, 1H), 6.84 (br, 2H). LRMS [M+H]=264.2

Example 27 2-methoxybenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-methoxypicolinonitrile

To a solution of 3,5-dichloropicolinonitrile (1.0 eq.) in dimethylformamide (DMF) (0.5 M) was added sodium methoxide (1.5 eq.) and heatedto 75° C. After stirring for 14 hours, the reaction was diluted withethyl acetate and water. The organic layer was washed with saturatedaqueous NaHCO₃ three times, water twice, dried over anhydrous MgSO₄, andconcentrated en vacuo. The crude residue was purified by a COMBIFLASH®system (ISCO) using 15% ethyl acetate in hexane to give a mixture of twomethoxy regioisomers, one of which was the desired product. The mixturewas carried onto the next step without further purification.

Step 2: 2-methoxybenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-chloro-5-methoxypicolinonitrile (from step 1) (1.0 eq.) in toluene(0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium (5 mol %)and 2N aqueous potassium carbonate solution (2.0 eq.). The reaction washeated to 100° C. and stirred overnight. After cooling to ambienttemperature, the reaction content was diluted with 2% methanol indichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 50-100% ethylacetate in hexane to give 2-methoxybenzo[f][1,7]naphthyridin-5-amine.

Example 28 2-(benzyloxy)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-(benzyloxy)-5-bromopyridine

A solution of 5-bromopyridin-3-ol (1.0 eq.), benzyl bromide (1.2 eq.),and silver carbonate (1.3 eq.) in toluene (0.1 M) was heated to 50° C.and stirred for 18 hours. After cooling to room temperature, thereaction mixture was filtered, eluting with ethyl acetate. The filtratewas concentrated en vacuo into a residue that was purified by aCOMBIFLASH® system (ISCO) using 20% ethyl acetate in hexane to give3-(benzyloxy)-5-bromopyridine.

Step 2: 3-(benzyloxy)-5-bromopyridine 1-oxide

A solution of 3-(benzyloxy)-5-bromopyridine (from step 1) (1.0 eq.) andmeta-chloroperbenzoic acid (mCPBA) (4.0 eq.) in dichloromethane (0.1 M)was stirred at room temperature for 18 hours. The reaction was quenchedwith saturated aqueous NaHCO₃ solution and extracted withdichloromethane three times. The combined organic layers were dried overanhydrous MgSO₄ and concentrated en vacuo. The crude residue waspurified by a COMBIFLASH® system (ISCO) using 0-100% ethyl acetate inhexane to give 3-(benzyloxy)-5-bromopyridine 1-oxide.

Step 3: 5-(benzyloxy)-3-bromopicolinonitrile

To a solution of 53-(benzyloxy)-5-bromopyridine 1-oxide (from step 2)(1.0 eq.) in acetonitrile (0.2 M) was added trimethylsilyl cyanide(TMSCN) (4.0 eq.) and triethylamine (3.0 eq.). The reaction was heatedat 100° C. overnight. After cooling to room temperature, the solvent wasconcentrated en vacuo, and the residue was purified by a COMBIFLASH®system (ISCO) using 0-40% ethyl acetate in hexane to give a mixture oftwo benzoxy regioisomers, one of which was the desired product. Themixture was carried onto the next step without further purification.

Step 4: 2-(benzyloxy)benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 5-(benzyloxy)-3-bromopicolinonitrile (from step 3) (1.0 eq.) intoluene (0.44 M) was mixed with tetrakis(triphenyl-phosphine)palladium(5 mol %) and 2N aqueous potassium carbonate solution (2.0 eq.). Thereaction was heated to 100° C. and stirred overnight. After cooling toambient temperature, the reaction content was diluted with 2% methanolin dichloromethane and water. The two phases were separated, and theaqueous layer was extracted twice with 2% methanol in dichloromethane.The combined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vacuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 50-100% ethylacetate in hexane to give2-(benzyloxy)benzo[f][1,7]naphthyridin-5-amine. ¹H NMR (acetone d-6): δ8.36 (s, 1H), 7.86 (s, 1H), 7.59-7.56 (d, 2H), 7.46-7.42 (dd, 2H),7.40-7.37 (d, 1H), 7.20-7.15 (dd, 1H), 7.12-7.09 (d, 1H), 6.88-6.86 (d,1H), 6.77-6.73 (dd, 1H), 5.51 (s, 2H), 4.74 (br, 2H). LRMS [M+H]=302.3.

Example 29 2-vinylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-vinylpicolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (3.4 eq.) in toluene/ethanol (2:1, 0.04 M) wasstirred at 95° C. overnight. After cooling to ambient temperature, thereaction content was diluted with ethyl acetate and water. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vacuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to give a white solid.

Step 2: 2-vinylbenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-chloro-5-vinylpicolinonitrile (from step 1) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane togive 2-vinylbenzo[f][1,7]naphthyridin-5-amine as a yellow solid. ¹H NMR(methanol-d4-CDCl₃): δ 8.87 (d, 1H), 8.69 (d, 1H), 8.28 (d, 1H),7.49-7.58 (m, 2H), 7.32 (dt, 1H), 6.90 (dd, 1H), 6.09 (d, 1H), 5.54 (d,1H). LRMS [M+H]=222.1.

Example 30 2-ethylbenzo[f][1,7]naphthyridin-5-amine

To a solution of 2-vinylbenzo[f][1,7]naphthyridin-5-amine (Example 29)in ethyl acetate/methanol (1:4, 0.05 M) was added 10% wt palladium oncarbon (0.2 eq.). Hydrogen gas was introduced via a balloon, and thereaction was stirred for 3 hours. The mixture was filtered through a padof celite, washing with dichloromethane. The filtrate was concentrateden vacuo and purified by a COMBIFLASH® system (ISCO) using 0-80% ethylacetate in hexane to give 2-ethylbenzo[f][1,7]naphthyridin-5-amine as asolid. ¹H NMR (methanol-d4): δ 8.78-8.81 (m, 2H), 8.45 (d, 1H),7.55-7.63 (m, 2H), 7.35-7.40 (m, 1H), 2.97 (q, 2H), 1.43 (t, 2H). LRMS[M+H]=224.1.

Example 31 2-phenylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-phenylpicolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),4,4,5,5-tetramethyl-2-phenyl-1,3,2-dioxaborolane (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (3.4 eq.) in toluene/ethanol (2:1, 0.04 M) wasstirred at 100° C. for 2 hours, then 80° C. for 4 hours. After coolingto ambient temperature, the reaction content was diluted with ethylacetate and water. The two phases were separated, and the aqueous layerwas extracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envacuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give awhite solid.

Step 2: 2-phenylbenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-chloro-5-phenylpicolinonitrile (from step 1) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude product was purified by flash chromatography ona COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give2-phenylbenzo[f][1,7]naphthyridin-5-amine as a white solid. ¹H NMR(dmso-d6): δ 9.13 (d, 1H), 9.03 (d, 1H), 8.56 (d, 1H), 7.98 (d, 2H),7.43-7.56 (m, 5H), 7.27 (m, 1H), 7.13 (bs, 2H). LRMS [M+H]=272.2.

Example 32

(E)-2-styrylbenzo[f][1,7]naphthyridin-5-amine

Step 1: (E)-3-chloro-5-styrylpicolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),(E)-4,4,5,5-tetramethyl-2-styryl-1,3,2-dioxaborolane (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (3.4 eq.) in toluene/ethanol (2:1, 0.04 M) wasstirred at 100° C. for 2 hours, then 80° C. for 4 hours. After coolingto ambient temperature, the reaction content was diluted with ethylacetate and water. The two phases were separated, and the aqueous layerwas extracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envacuo. The crude product was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give awhite solid.

Step 2: (E)-2-styrylbenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and (E)-3-chloro-5-styrylpicolinonitrile (from step 1) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude product was purified by flash chromatography ona COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give(E)-2-styrylbenzo[f][1,7]naphthyridin-5-amine as a brown solid. ¹H NMR(dmso-d6): δ 9.22 (d, 1H), 9.06 (d, 1H), 8.51 (d, 1H), 7.78 (d, 1H),7.66 (d, 2H), 7.46-7.56 (m, 3H), 7.70 (t, 2H), 7.26-7.32 (m, 2H), 7.08(bs, 2H). LRMS [M+H]=298.2.

Example 33 2-phenethylbenzo[f][1,7]naphthyridin-5-amine

To a solution of (E)-2-styrylbenzo[f][1,7]naphthyridin-5-amine (Example32) in ethyl acetate/methanol (1:4, 0.05 M) was added 10% wt palladiumon carbon (0.2 eq.). Hydrogen gas was introduced via a balloon, and thereaction was stirred for 3 hours. The mixture was filtered through a padof celite, washing with dichloromethane. The filtrate was concentrateden vacua and the crude product was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give2-phenethylbenzo[f][1,7]naphthyridin-5-amine as a yellow solid. ¹H NMR(CDCl₃): δ 8.54 (d, 1H), 8.32 (d, 1H), 8.10 (dd, 1H), 7.63 (dd, 1H),7.51 (m, 1H), 7.03-7.32 (m, 6H), 6.16 (bs, 2H), 3.11 (t, 2H), 2.97 (t,2H). LRMS [M+H]=300.1.

Example 34

(E)-2-(3-methoxyprop-1-enyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: (E)-3-chloro-5-(3-methoxyprop-1-enyl)picolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),(E)-2-(3-methoxyprop-1-enyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1.0 eq.), tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2Naqueous sodium carbonate solution (3.4 eq.) in toluene/ethanol (2:1,0.04 M) was stirred at 100° C. for 2 hours, then 80° C. for 4 hours.After cooling to ambient temperature, the reaction content was dilutedwith ethyl acetate and water. The two phases were separated, and theaqueous layer was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vacua. The crude product was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-80% ethyl acetatein hexane to give (E)-3-chloro-5-(3-methoxyprop-1-enyl)picolinonitrileas a white solid.

Step 2: (E)-2-(3-methoxyprop-1-enyl)benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and (E)-3-chloro-5-(3-methoxyprop-1-enyl)picolinonitrile (from step 1)(1.0 eq.), tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2Naqueous sodium carbonate solution (2.0 eq.) in toluene/ethanol (2:1,0.03 M) was stirred at 100° C. overnight. After cooling to ambienttemperature, the reaction content was diluted with methanol. Theinsoluble solids were filtered off, and the filtrate was concentrated envacuo to obtain a crude residue. The crude product was purified by flashchromatography on a COMBIFLASH®, system (ISCO) using 0-80% ethyl acetatein hexane to give(E)-2-(3-methoxyprop-1-enyl)benzo[f][1,7]naphthyridin-5-amine as ayellow solid. ¹H NMR (dmso-d6): δ 9.24 (d, 1H), 9.18 (d, 1H), 8.54 (d,1H), 7.52-7.58 (m, 2H), 7.31 (m, 1H), 7.11 (bs, 2H), 6.86-7.00 (m, 2H),4.18 (d, 2H), 3.36 (s, 3H). LRMS [M+H]=266.2.

Example 35 2-(3-methoxypropyl)benzo[f][1,7]naphthyridin-5-amine

To a solution of(E)-2-(3-methoxyprop-1-enyl)benzo[f][1,7]naphthyridin-5-amine (Example34) in ethyl acetate/methanol (1:4, 0.05 M) was added 10% wt palladiumon carbon (0.2 eq.). Hydrogen gas was introduced via a balloon, and thereaction was stirred for 3 hours. The mixture was filtered through a padof celite, washing with dichloromethane. The filtrate was concentrateden vacuo and the crude product was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give2-(3-methoxypropyl)benzo[f][1,7]naphthyridin-5-amine as a white solid.¹H NMR (CDCl₃): δ 8.64 (d, 1H), 8.46 (d, 1H), 8.19 (d, 1H), 7.66 (d,1H), 7.53 (m, 1H), 7.31 (m, 1H), 6.56 (bs, 2H), 3.37 (t, 2H), 3.31 (s,3H), 2.91 (t, 2H), 1.93-2.00 (m, 2H). LRMS [M+H]=268.1.

Example 36 2-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-(prop-1-en-2-yl)picolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (3.4 eq.) in toluene/ethanol (2:1, 0.04 M) wasstirred at 100° C. for 2 hours, then 80° C. for 4 hours. After coolingto ambient temperature, the reaction content was diluted with ethylacetate and water. The two phases were separated, and the aqueous layerwas extracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envacuo. The crude product was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give3-chloro-5-(prop-1-en-2-yl)picolinonitrile as a white solid.

Step 2: 2-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-chloro-5-(prop-1-en-2-yl)picolinonitrile (from step 1) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude product was purified by flash chromatography ona COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give2-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine as a white solid. ¹HNMR (dmso-d6): δ 9.03 (d, 1H), 8.96 (d, 1H), 8.55 (d, 1H), 7.47-7.53 (m,2H), 7.25 (m, 1H), 7.07 (bs, 2H) 5.80 (s, 1H), 5.36 (s, 1H), 2.27 (s,3H). LRMS [M+H]=236.2.

Example 37 2-isopropylbenzo[f][1,7]naphthyridin-5-amine

To a solution of 2-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine(Example 36) in ethyl acetate/methanol (1:4, 0.05 M) was added 10% wtpalladium on carbon (0.2 eq.). Hydrogen gas was introduced via aballoon, and the reaction was stirred for 3 hours. The mixture wasfiltered through a pad of celite, washing with dichloromethane. Thefiltrate was concentrated en vacuo and the crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-80% ethylacetate in hexane to give 2-isopropylbenzo[f][1,7]naphthyridin-5-amineas a yellow solid. ¹H NMR (CDCl₃): δ 8.69 (d, 1H), 8.49 (d, 1H), 8.25(dd, 1H), 7.65 (dd, 1H), 7.53 (m, 1H), 7.31 (m, 1H), 6.02 (bs, 2H), 3.15(septet, 1H), 1.37 (d, 6H). LRMS [M+H]=238.2.

Example 38 1-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 5-bromo-2-chloro-4-methylpyridine 1-oxide

A solution of 5-bromo-2-chloro-4-methylpyridine (1.0 eq.) andmeta-chloroperbenzoic acid (mCPBA) (2.5 eq.) in chloroform (0.1 M) wasstirred at 50° C. overnight. After cooling to room temperature, Ca(OH)₂(2.5 eq.) was added to the reaction mixture. The precipitate wasfiltered and washed with 5% methanol in dichloromethane and ethylacetate. The filtrate was washed with saturated aqueous Na₂S₂O₃ solutionand saturated aqueous NaHCO₃ solution. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated en vacuointo a pale solid that was carried onto the next step without furtherpurification.

Step 2: 3-bromo-6-chloro-4-methylpicolinonitrile

To a solution of 5-bromo-2-chloro-4-methylpyridine 1-oxide (from step 1)(1.0 eq.) in acetonitrile (0.2 M) was added TMSCN (4.0 eq.) andtriethylamine (3.0 eq.). The reaction was heated at 100° C. overnight.After cooling to room temperature, the solvent was concentrated envacuo, and the residue was purified by a COMBIFLASH® system (ISCO) using0-50% ethyl acetate in hexane to give3-bromo-6-chloro-4-methylpicolinonitrile.

Step 3: 3-chloro-1-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of 2-(tert-butoxycarbonylamino)phenylboronic acid (1.0 eq.)and 3-bromo-6-chloro-4-methylpicolinonitrile (from step 2) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude product was purified by flash chromatography ona COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give3-chloro-1-methylbenzo[f][1,7]naphthyridin-5-amine as a white solid. ¹HNMR (dmso-d6): δ 8.44 (d, 1H), 7.83 (s, 1H), 7.50-7.58 (m, 2H), 7.02(bs, 2H), 2.98 (s, 3H).

LRMS [M+H]=244.1.

Step 4: 1-methylbenzo[f][1,7]naphthyridin-5-amine

To a solution of 3-chloro-1-methylbenzo[f][1,7]naphthyridin-5-amine(from step 3) in ethyl acetate/methanol (1:2, 0.03 M) was added 10% wtpalladium on carbon (0.2 eq.). The reaction vessel was shaken on ahydrogen Parr apparatus under 50 psi of hydrogen overnight. The mixturewas filtered through a pad of celite, washing with dichloromethane. Thefiltrate was concentrated en vacuo and purified by a COMBIFLASH® system(ISCO) using 0-80% ethyl acetate in hexane to give1-methylbenzo[f][1,7]naphthyridin-5-amine as a white solid. ¹H NMR(CDCl₃): δ 8.63 (d, 1H), 8.44 (d, 1H), 7.71 (dd, 1H), 7.54 (m, 1H), 7.45(d, 1H), 7.30 (m, 1H), 6.20 (bs, 2H), 3.01 (s, 3H). LRMS [M+H]=210.1.

Example 40 pyrido[3,2-f][1,7]naphthyridin-6-amine

A solution of tert-butyl3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-ylcarbamate(1.0 eq.) and 3-bromopicolinonitrile (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude product was purified by flash chromatography ona COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to givepyrido[3,2-f][1,7]naphthyridin-6-amine as a white solid. ¹H NMR(dmso-d6): δ 9.14 (dd, 1H), 8.98 (dd, 1H), 8.90 (dd, 1H), 7.93 (dd, 1H),7.60 (bs, 2H), 7.30 (dd, 1H). LRMS [M+H]=197.

Example 41 2-ethyl-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 8-methyl-2-vinylbenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/step 2) (1.0 eq.) and 3-chloro-5-vinylpicolinonitrile(from Example 29/Step 1) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vacuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane togive 8-methyl-2-vinylbenzo[f][1,7]naphthyridin-5-amine as a yellowsolid.

Step 2: 2-ethyl-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a solution of 8-methyl-2-vinylbenzo[f][1,7]naphthyridin-5-amine (fromthe previous step) in ethyl acetate/methanol (1:4, 0.05 M) was added 10%wt palladium on carbon (0.2 eq.). Hydrogen gas was introduced via aballoon, and the reaction was stirred for 3 hours. The mixture wasfiltered through a pad of celite and washed with dichloromethane. Thefiltrate was concentrated en vaccuo and purified by a COMBIFLASH® system(ISCO) using 0-80% ethyl acetate in hexane to give2-ethyl-8-methylbenzo[f][1,7]naphthyridin-5-amine as an offwhite solid.¹H NMR (CDCl₃): δ 8.61 (d, 1H), 8.42 (d, 1H), 8.10 (d, 1H), 7.44 (s,1H), 7.12 (dd, 1H), 6.00 (bs, 2H), 2.84 (q, 2H), 2.45 (s, 3H), 1.33 (t,3H). LRMS [M+H]=238.1.

Example 42 (5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)methanol

Step 1: ethyl 5-chloro-6-cyanonicotinate

A solution of ethyl 5,6-dichloronicotinate (1 eq), zinc cyanide (0.75eq) and tetrakis(triphenyl-phosphine)palladium (0.10 eq.) in DMF (0.3 M)was degassed and then heated at 100° C. for 3 hours. Solvent was removeden vaccuo to obtain a crude residue. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-80% ethylacetate in hexane to give ethyl 5-chloro-6-cyanonicotinate as a whitesolid.

Step 2: ethyl 5-amino-8-methylbenzo[f][1,7]naphthyridine-2-carboxylate

A solution of tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/step 2) (1.0 eq.) and ethyl 5-chloro-6-cyanonicotinate(from the previous step) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction content was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vaccuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane togive ethyl 5-amino-8-methylbenzo[f][1,7]naphthyridine-2-carboxylate.

Step 3: 2-ethyl-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a stirred solution of ethyl5-amino-8-methylbenzo[f][1,7]naphthyridine-2-carboxylate (from theprevious step) in THF (0.2 M) cooled in an ice-water bath was added 1 Nsolution of super hydride in THF (10 eq.). Upon completion of thereaction the reaction was quenched with 1 N HCl, and extracted withEtOAc. Combined organic extracts were concentrated en vaccuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane togive (5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)methanol as a whitesolid. ¹H NMR (CDCl₃): δ 8.68 (d, 1H), 8.52 (d, 1H), 8.04 (d, 1H), 7.44(s, 1H), 7.12 (dd, 1H), 6.00 (bs, 2H), 4.90 (s, 2H), 2.45 (s, 3H). LRMS[M+H]=240.1

Example 43 8-methyl-2-propylbenzo[f][1,7]naphthyridin-5-amine

Step 1: (E)-3-chloro-5-(prop-1-enyl)picolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),(E)-4,4,5,5-tetramethyl-2-(prop-1-enyl)-1,3,2-dioxaborolane (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (3.4 eq.) in toluene/ethanol (2:1, 0.04 M) wasstirred at 95° C. overnight. After cooling to ambient temperature, thereaction content was diluted with ethyl acetate and water. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo to obtain a cruderesidue. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane to give awhite solid (E)-3-chloro-5-(prop-1-enyl)picolinonitrile.

Step 2: (E)-8-methyl-2-(prop-1-enyl)benzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.0 eq.) and(E)-3-chloro-5-(prop-1-enyl)picolinonitrile (from the previous step)(1.0 eq.), tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2Naqueous sodium carbonate solution (2.0 eq.) in toluene/ethanol (2:1,0.03 M) was stirred at 100° C. overnight. After cooling to ambienttemperature, the reaction content was diluted with methanol. Theinsoluble solids were filtered off, and the filtrate was concentrated envaccuo to obtain a crude residue. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-80% ethylacetate in hexane to give(E)-8-methyl-2-(prop-1-enyl)benzo[f][1,7]naphthyridin-5-amine as ayellow solid.

Step 3: 8-methyl-2-propylbenzo[f][1,7]naphthyridin-5-amine

To a solution of(E)-8-methyl-2-(prop-1-enyl)benzo[f][1,7]naphthyridin-5-amine (from theprevious step) in ethyl acetate/methanol (1:4, 0.05 M) was added 10% wtpalladium on carbon (0.2 eq.). Hydrogen gas was introduced via aballoon, and the reaction was stirred for 3 hours. The mixture wasfiltered through a pad of celite, washing with dichloromethane. Thefiltrate was concentrated en vaccuo and purified by a COMBIFLASH® system(ISCO) using 0-80% ethyl acetate in hexane to give8-methyl-2-propylbenzo[f][1,7]naphthyridin-5-amine as offwhite solid. ¹HNMR (CDCl₃): δ 8.59 (d, 1H), 8.41 (d, 1H), 8.10 (d, 1H), 7.43 (s, 1H),7.13 (dd, 1H), 5.94 (bs, 2H), 2.78 (t, 2H), 2.44 (s, 3H), 1.75 (m, 2H),0.95 (t, 3H). LRMS [M+H]=252.1

Example 442-(2-(1H-indol-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 5-((triethylsilyl)ethynyl)-1H-indole

To a scintillation vial was added -iodo-1H-indole (1.1 eq.),triethyl(ethynyl)silane (1 eq.), triethylamine (5 eq.), and anhydrousDMF (0.2 M). Vacuumed and nitrogen flushed for three times. CuI (0.1eq.) and bis(triphenylphosphine)dichloro-palladium(II) (0.1 eq) wereadded. The vial was sealed and heated at 60° C. overnight. Uponcompletion of the reaction as monitored by TLC, the content of the vialwas loaded onto a silica gel column pretreated with hexanes. Column waswashed with hexanes and diethylether until all eluents containingproduct were collected. Carefully distill off hexanes and ether usingrotary evaporator with minim heating afforded product5-((triethylsilyl)ethynyl)-1H-indole as colorless oil, which was carrieddirectly on to the next step.

Step 2: 5-ethynyl-1H-indole

To a stirred solution of 5-((triethylsilyl)ethynyl)-1H-indole (from theprevious step) in THF (0.2 M) cooled at 0° C. was treated with asolution (0.5 eq.) of tetrabutylammonium fluoride in a dropwise fashion.The reaction mixture turned black and was continued to stir for 30minutes before warming up to rt. TLC showed full conversion. Thereaction was quenched with water and was extracted with diethylether.Combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated using rotary evaporator with minim heating. Chromatography(silica gel, diethylether) afforded the product 5-ethynyl-1H-indole ascolorless oil.

Step 3: 5-((1H-indol-5-yl)ethynyl)-3-chloropicolinonitrile

To a round bottom flask capped with septa was added 5-ethynyl-1H-indole(from the previous step) (1.1 eq), 3,5-dichloropicolinonitrile (1 eq.),triethylamine (5 eq.), and anhydrous DMF (0.2 M). Vacuumed and nitrogenflushed for three times. CuI (0.05 eq.) andbis(triphenylphosphine)dichloro-palladium(II) (0.05 eq) were added. Theseptum was replaced with a refluxing condenser and the flask was heatedat 60° C. overnight under nitrogen atmosphere. Upon completion of thereaction as monitored by TLC, the content of the flask was loaded onto alarge silica gel column pretreated with hexanes. Flash chromatography(silica gel, hexanes:EtOAc (1:4%)) afforded the product5-((1H-indol-5-yl)ethynyl)-3-chloropicolinonitrile.

Step 4:2-((1H-indol-5-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a round bottom flask with refluxing condenser were added5-((1H-indol-5-yl)ethynyl)-3-chloropicolinonitrile (from the previousstep) (1 eq.), tert-butyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate (fromExample 5/Step 2) (1.25 eq.), K₃PO₄ (2 eq.),tris(dibenzylideneacetone)dipalladium(0) (0.05 eq.), and2-Dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (0.1 eq.). n-Butanol andwater (5:2, 0.2 M) were added, and the content were degassed (vacuumfollowed by nitrogen flush) for three times. The reaction mixture wasstirred vigorously under nitrogen at 100° C. overnight in an oil bath.The content were cooled down and were taken up in 200 mL of waterfollowed by extraction with methylene chloride. Combined organic layerswere dried (Na₂SO₄) and concentrated. Flash chromatography (silica gel,0-50% EtOAc in CH₂Cl₂) afforded the product2-((1H-indol-5-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine asa yellow solid.

Step 5:2-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a round bottom flask was added2-((1H-indol-5-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) (1 eq.) with a stirring bar. Ethanol andmethylene chloride (1:2, 0.2 M) were added, followed by palladium incarbon (activated powder, wet, 10% on carbon, 0.1 eq.). The content werevacuumed followed by hydrogen flush for three times. The reactionmixture was stirred vigorously under hydrogen balloon at roomtemperature overnight. Afterwards the reaction mixture was filteredthrough a celite pad, and the celite pad was washed subsequently withmethylene chloride and EtOAc until the filtrate had no UV absorption.Combined organic washes were concentrated. Flash chromatography (silicagel, 0-50% EtOAc in CH₂Cl₂) afforded the product2-(2-(2,3-Dihydrobenzofuran-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amineas a yellow solid. ¹H NMR (CDCl₃): δ 8.54 (d, 1H), 8.34 (d, 1H), 8.28(s, 1H), 7.99 (d, 1H), 7.64-7.56 (m, 1H), 7.50-7.35 (m, 1H), 7.24 (d,1H), 7.12 (t, 1H), 7.08 (dd, 1H), 6.92 (dd, 1H), 6.41 (s, 1H), 6.01 (bs,2H), 3.16-3.12 (m, 2H), 3.10-3.05 (m, 2H), 2.43 (s, 3H). LRMS[M+H]=353.2

Example 452-(4-ethoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-((4-ethoxyphenyl)ethynyl)picolinonitrile

To a round bottom flask capped with septa was added1-ethoxy-4-ethynylbenzene (1.1 eq), 3,5-dichloropicolinonitrile (1 eq.),triethylamine (5 eq.), and anhydrous DMF (0.2 M). Vacuumed and nitrogenflushed for three times. CuI (0.05 eq.) andbis(triphenylphosphine)dichloro-palladium(II) (0.05 eq) were added. Theseptum was replaced with a refluxing condenser and the flask was heatedat 60° C. overnight under nitrogen atmosphere. Upon completion of thereaction as monitored by TLC, the content of the flask was loaded onto alarge silica gel column pretreated with hexanes. Flash chromatography(silica gel, hexanes:EtOAc (1:4%)) afforded the product3-chloro-5-((4-ethoxyphenyl)ethynyl)picolinonitrile.

Step 2:2-((4-ethoxyphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a round bottom flask with refluxing condenser were added3-chloro-5-((4-ethoxyphenyl)ethynyl)picolinonitrile (from the previousstep) (1 eq.), tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.25 eq.), K₃PO₄ (2 eq.),tris(dibenzylideneacetone)dipalladium(0) (0.05 eq.), and2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (0.1 eq.). n-Butanol andwater (5:2, 0.2 M) were added, and the content were degassed (vacuumfollowed by nitrogen flush) for three times. The reaction mixture wasstirred vigorously under nitrogen at 100° C. overnight in an oil bath.The content were cooled down and were taken up in 200 mL of waterfollowed by extraction with methylene chloride. Combined organic layerswere dried (Na₂SO₄) and concentrated. Flash chromatography (silica gel,0-50% EtOAc in CH₂Cl₂) afforded the product2-((4-ethoxyphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine.

Step 3: 2-(4-ethoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a round bottom flask was added2-((4-ethoxyphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) (1 eq.) with a stirring bar. Ethanol andmethylene chloride (1:2, 0.2 M) were added, followed by palladium incarbon (activated powder, wet, 10% on carbon, 0.1 eq.). The contentswere degassed under vacuum followed by hydrogen flush (three times). Thereaction mixture was stirred vigorously under hydrogen balloon at roomtemperature overnight. Afterwards the reaction mixture was filteredthrough a celite pad, and the celite pad was washed subsequently withmethylene chloride and EtOAc until the filtrate had no UV absorption.Combined organic washes were concentrated. Flash chromatography (silicagel, 0-50% EtOAc in CH₂Cl₂) afforded the product as a yellow solid.Further recrystallization using toluene afforded product2-(4-ethoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine as awhite fine crystal. ¹H NMR (CDCl₃): δ 8.52 (d, 1H), 8.30 (d, 1H), 8.10(d, 1H), 7.46 (s, 1H), 7.12 (dd, 1H), 7.06 (d, 2H), 6.75 (d, 2H), 5.95(bs, 2H), 3.93 (q, 2H), 3.11-3.05 (dd, 2H), 2.95-2.90 (dd, 2H), 2.44 (s,3H), 1.33 (t, 3H). LRMS [M+H]=358.2

Example 468-methyl-2-(4-phenoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-((4-phenoxyphenyl)ethynyl)picolinonitrile

3-Chloro-5-((4-phenoxyphenyl)ethynyl)picolinonitrile was prepared from1-ethynyl-4-phenoxybenzene (commercially available) following theprocedures described for Example 45, step 1.

Step 2:8-methyl-2-((4-phenoxyphenyl)ethynyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-((4-phenoxyphenyl)ethynyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from 3-chloro-5-((4-phenoxyphenyl)ethynyl)picolinonitrile(from the previous step) following the procedures described for Example45, step 2.

Step 3: 8-methyl-2-(4-phenoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-phenoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine wasprepared from8-methyl-2-((4-phenoxyphenyl)ethynyl)benzo[f][1,7]naphthyridin-5-amine(from the previous step) following the procedures described for Example45, step 3. ¹H NMR (CDCl₃): δ 8.54 (d, 1H), 8.30 (d, 1H), 8.01 (d, 1H),7.45 (s, 1H), 7.25-7.20 (m, 2H), 7.12 (dd, 1H), 7.07-6.84 (m, 8H), 6.00(bs, 2H), 3.13-3.08 (dd, 2H), 2.99-2.94 (dd, 2H), 2.44 (s, 3H). LRMS[M+H]=406.2

Example 47 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: ((2,4-dimethylphenyl)ethynyl)triethylsilane

((2,4-Dimethylphenyl)ethynyl)triethylsilane was prepared from1-iodo-2,4-dimethylbenzene (commercially available) following theprocedures described for Example 44, step 1.

Step 2: 1-ethynyl-2,4-dimethylbenzene

1-Ethynyl-2,4-dimethylbenzene was prepared from((2,4-dimethylphenyl)ethynyl)triethylsilane (from the previous step)following the procedures described for Example 44, step 2.

Step 3: 3-chloro-5-((2,4-dimethylphenyl)ethynyl)picolinonitrile

3-Chloro-5-((2,4-dimethylphenyl)ethynyl)picolinonitrile was preparedfrom 1-ethynyl-2,4-dimethylbenzene (from the previous step) followingthe procedures described for Example 44, step 3.

Step 4: 2-((2,4-dimethylphenyl)ethynyl)benzo[f][1,7]naphthyridin-5-amine

2-((2,4-Dimethylphenyl)ethynyl)benzo[f][1,7]naphthyridin-5-amine wasprepared from 3-chloro-5-((2,4-dimethylphenyl)ethynyl)-picolinonitrile(from the previous step) following the procedures described for Example44, step 4.

Step 5: 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine

2-(2,4-Dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine was preparedfrom 2-((2,4-dimethylphenyl)ethynyl)benzo[f][1,7]naphthyridin-5-amine(from the previous step) following the procedures described for Example44, step 5. ¹H NMR (CDCl₃): δ 8.60 (d, 1H), 8.33 (d, 1H), 8.14 (d, 1H),7.67 (d, 1H), 7.54 (t, 1H), 7.31 (t, 1H), 6.96-6.86 (m, 3H), 6.29 (bs,2H), 3.04-3.10 (dd, 2H), 2.97-2.91 (dd, 2H), 2.24 (s, 3H), 2.20 (s, 3H).LRMS [M+H]=328.2.

Example 482-(2,4-dimethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1:2-((2,4-dimethylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-((2,4-Dimethylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from3-chloro-5-((2,4-dimethylphenyl)ethynyl)picolinonitrile (from Example47/Step 3) and tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) following the procedures described for Example44, step 4.

Step 2:2-(2,4-dimethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(2,4-Dimethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine wasprepared from 1-ethynyl-4-phenoxybenzene (from the previous step)following the procedures described for Example 44, step 5. ¹H NMR(CDCl₃): δ 8.56 (d, 1H), 8.28 (d, 1H), 8.00 (d, 1H), 7.46 (s, 1H), 7.14(dd, 1H), 6.95-6.85 (m, 3H), 6.26 (bs, 2H), 3.08-3.02 (dd, 2H),2.96-2.90 (dd, 2H), 2.45 (s, 3H), 2.23 (s, 3H), 2.19 (s, 3H). LRMS[M+H]=342.2

Example 492-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile

3-Chloro-5-((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile wasprepared from 1-ethynyl-4-methoxy-2-methylbenzene (commerciallyavailable) following the procedure described for Example 44/Step 3.

Step 2:2-((4-methoxy-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-((4-Methoxy-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from3-chloro-5-(4-methoxy-2-methylphenethyl)picolinonitrile (from theprevious step) following the procedures described for Example 44, step4.

Step 3:2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-Methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from2-((4-methoxy-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) following the procedures described for Example44, step 5. ¹H NMR (CDCl₃): δ 8.53 (d, 1H), 8.29 (d, 1H), 8.01 (d, 1H),7.44 (s, 1H), 7.12 (dd, 1H), 6.93 (d, 1H), 6.67 (d, 1H), 6.60 (dd, 1H),5.93 (bs, 2H), 3.70 (s, 3H), 3.05-3.00 (dd, 2H), 2.93-2.88 (dd, 2H),2.44 (s, 3H), 2.19 (s, 3H). LRMS [M+H]=358.2

Example 504-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol

To a stirred solution of2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(Example 49) in methylene chloride (0.2 M) in an ice-water bath wasadded 1 N solution of BBr₃ (2 eq) in CH₂Cl₂ in a drop-wise fashion. In30 minutes the reaction was quenched with methanol and was concentrateden vaccuo to obtain a crude residue. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-20% methanolin dichloromethane to give4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenolas a white solid. ¹H NMR (DMSO-d₆): δ 8.99 (s, 1H), 8.75 (d, 1H), 8.60(d, 1H), 8.27 (d, 1H), 7.28 (s, 1H), 7.09 (dd, 1H), 6.99 (bs, 2H), 6.88(d, 1H), 6.49 (d, 1H), 6.42 (dd, 1H), 3.02-2.96 (dd, 2H), 2.86-2.81 (dd,2H), 2.38 (s, 3H), 2.13 (s, 3H). LRMS [M+H]=344.2

Example 512-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: ((2,3-dihydrobenzofuran-5-yl)ethynyl)triethylsilane

((2,3-Dihydrobenzofuran-5-yl)ethynyl)triethylsilane was prepared from5-iodo-2,3-dihydrobenzofuran (commercially available) following theprocedures described for Example 44, step 1.

Step 2: 5-ethynyl-2,3-dihydrobenzofuran

5-Ethynyl-2,3-dihydrobenzofuran was prepared from((2,3-dihydrobenzofuran-5-yl)ethynyl)triethylsilane (from the previousstep) following the procedures described for Example 44/Step 2.

Step 3: 3-chloro-5-((2,3-dihydrobenzofuran-5-yl)ethynyl)picolinonitrile

3-Chloro-5-((2,3-dihydrobenzofuran-5-yl)ethynyl)picolinonitrile wasprepared from 5-ethynyl-2,3-dihydrobenzofuran (from the previous step)following the procedures described for Example 44/Step 3.

Step 4:2-((2,3-dihydrobenzofuran-5-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-((2,3-Dihydrobenzofuran-5-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from3-chloro-5-(4-methoxy-2-methylphenethyl)picolinonitrile (from theprevious step) following the procedures described for Example 44/Step 4.

Step 5:2-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(2-(2,3-Dihydrobenzofuran-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from2-((2,3-dihydrobenzofuran-5-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) following the procedures described for Example44/Step 5. ¹H NMR (CDCl₃): δ 8.62 (d, 1H), 8.40 (d, 1H), 8.11 (d, 1H),7.53 (s, 1H), 7.21 (dd, 1H), 6.99 (s, 1H), 6.95 (dd, 1H), 6.74 (d, 1H),6.05 (bs, 2H), 4.57 (t, 2H), 3.19-3.13 (m, 4H), 3.03-2.98 (dd, 2H), 2.54(s, 3H). LRMS [M+H]=356.2

Example 52 2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethanol

Step 1: (Z)-3-chloro-5-(2-ethoxyvinyl)picolinonitrile

(Z)-3-Chloro-5-(2-ethoxyvinyl)picolinonitrile was prepared from(Z)-2-(2-ethoxyvinyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(commercially available) following the procedures described for Example43/Step 1.

Step 2: (Z)-2-(2-ethoxyvinyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

(Z)-2-(2-Ethoxyvinyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine wasprepared from (Z)-3-chloro-5-(2-ethoxyvinyl)picolinonitrile (from theprevious step) following the procedures described for Example 43/Step 2.

Step 3: 2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethanol

A solution of(Z)-2-(2-ethoxyvinyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine (fromthe previous step) in a mixture of 2:5 conc. HCl and dioxane (0.1 M) washeated at 60° C. overnight. Upon cooling down to rt, the reactionmixture was treated with excess NaHCO₃ saturated solution, followed byextraction with EtOAc. Combined organic extracts were concentrated andwas taken up in THF (0.2 M), and was treated with 1 N super hydridesolution in THF (10 eq.) at 0° C. The reaction mixture was allowed towarm to room temperature and stirred overnight. The reaction was workedup following the procedures described for Example 42/Step 3, to afford2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethanol as whitesolid. ¹H NMR (CDCl₃): δ 8.61 (d, 1H), 8.47 (d, 1H), 8.01 (d, 1H), 7.41(s, 1H), 7.10 (d, 1H), 6.40 (s, 1H), 6.01 (bs, 2H), 4.01 (t, 2H), 3.06(t, 2H), 2.43 (s, 3H). LRMS [M+H]=254.1

Example 533-methyl-9-phenyl-9,10-dihydrobenzo[f]furo[2,3-b][1,7]naphthyridin-6-amine

Step 1: 5-bromo-2-chloro-3-methylpyridine 1-oxide

5-Bromo-2-chloro-3-methylpyridine 1-oxide was prepared from5-bromo-2-chloro-3-methylpyridine (commercially available) following theprocedures described for Example 19/Step 1.

Step 2: 3-bromo-6-chloro-5-methylpicolinonitrile

3-Bromo-6-chloro-5-methylpicolinonitrile was prepared from(Z)-3-chloro-5-(2-ethoxyvinyl)picolinonitrile (from the previous step)following the procedures described for Example 19/Step 2.

Step 3: 3-bromo-6-chloro-5-(2-hydroxy-2-phenylethyl)picolinonitrile

A solution of 3-bromo-6-chloro-5-methylpicolinonitrile (from theprevious step) in THF (0.2 M) was cooled to −78° C. LDA (2N solution, 2eq) was added dropwise. The reaction was kept stirring at −78° C. for 1hour, followed by addition of benzaldehyde (1 eq). The reaction was keptstirring at −78° C. for another 30 minutes before allowing it to slowlywarm to room temperature. The reaction was quenched with sat. NH₄Cl andextracted with EtOAc. Combined organic washes were concentrated. Flashchromatography (silica gel, 20-50% EtOAc in hexanes) afforded theproduct 3-bromo-6-chloro-5-(2-hydroxy-2-phenylethyl)picolinonitrile as ayellow solid.

Step 4:3-methyl-9-phenyl-9,10-dihydrobenzo[f]furo[2,3-b][1,7]naphthyridin-6-amine

3-Methyl-9-phenyl-9,10-dihydrobenzo[f]furo[2,3-b][1,7]naphthyridin-6-aminewas prepared from 3-bromo-6-chloro-5-methylpicolinonitrile (from theprevious step) following the procedures described for Example 44/Step 4.¹H NMR (CDCl₃): δ 8.45 (s, 1H), 7.98 (d, 1H), 7.45 (s, 1H), 7.40-7.28(m, 5H), 7.12 (d, 1H), 5.93 (t, 1H), 5.93 (brs, 2H), 3.86 (dd, 1H), 3.40(dd, 1H), 2.44 (s, 3H). LRMS [M+H]=328.1

Example 54 8-methylbenzo[f][1,7]naphthyridine-2,5-diamine

Step 1: tert-butyl 5,6-dichloropyridin-3-ylcarbamate

To a solution of 5,6-dichloropyridin-3-amine (commercially available) inTHF (0.2 M) stirred at 0° C. was added (BOC)₂O (1.2 eq). The reactionmixture was heated at 40° C. until full conversion as monitored by TLC.The reaction mixture was then concentrated. Flash chromatography (silicagel, 20-50% EtOAc in hexanes) of the crude afforded tert-butyl5,6-dichloropyridin-3-ylcarbamate.

Step 2: tert-butyl 5-chloro-6-cyanopyridin-3-ylcarbamate

Tert-butyl 5-chloro-6-cyanopyridin-3-ylcarbamate was prepared fromtert-butyl 5,6-dichloropyridin-3-ylcarbamate (from the previous step)following the procedures described for Example 42/Step 1.

Step 3: 8-methylbenzo[f][1,7]naphthyridine-2,5-diamine

8-methylbenzo[f][1,7]naphthyridine-2,5-diamine was prepared (as minorproduct) together with tert-butyl5-amino-8-methylbenzo[f][1,7]naphthyridin-2-ylcarbamate (as majorproduct) from tert-butyl 5-chloro-6-cyanopyridin-3-ylcarbamate (from theprevious step) following the procedures described for Example 5/Step 2.¹H NMR (DMSO-d₆): δ 10.11 (s, 1H), 9.02 (s, 1H), 8.82 (d, 1H), 8.06 (d,1H), 7.34 (s, 1H), 7.15 (dd, 1H), 6.99 (s, 2H), 2.44 (s, 3H). LRMS[M+H]=225.1

Example 55 1-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)propan-2-ol

Step 1: 3-bromo-5-methylpicolinonitrile

3-Bromo-5-methylpicolinonitrile was prepared from2,3-dibromo-5-methylpyridine (commercially available) following theprocedures described for Example 42/Step 1.

Step 2: 3-bromo-5-(2-hydroxypropyl)picolinonitrile

3-Bromo-5-(2-hydroxypropyl)picolinonitrile was prepared from3-bromo-5-methylpicolinonitrile (from the previous step) andacetaldehyde following the procedures described for Example 53/Step 3.

Step 3: 1-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)propan-2-ol

1-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)propan-2-ol wasprepared from 3-bromo-5-(2-hydroxypropyl)picolinonitrile (from theprevious step) following the procedures described for Example 53, step4. ¹H NMR (methanol-d₄): δ 8.72 (d, 1H), 8.68 (d, 1H), 8.24 (d, 1H),7.38 (s, 1H), 7.18 (dd, 1H), 4.16-4.07 (m, 1H), 3.05-2.99 (m, 2H),2.97-2.90 (m, 2H), 2.47 (s, 3H), 1.28 (d, 3H). LRMS [M+H]=268.1

Example 562-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)acetonitrile

Step 1: 2,3-dichloro-5-((methoxymethoxy)methyl)pyridine

To a stirred solution of (5,6-dichloropyridin-3-yl)methanol(commercially available) in CH₂Cl₂ (0.2 M) at 0° C. was addedtriethylamine (3 eq.) and chloro(methoxy)methane (2 eq.). After stirringat 0° C. for 3 hours the reaction mixture was concentrated and the crudewas purified by chromatography (silica gel, 20-50% EtOAc in hexanes) toafford 2,3-dichloro-5-((methoxymethoxy)methyl)pyridine as a colorlessoil.

Step 2: 3-chloro-5-((methoxymethoxy)methyl)picolinonitrile

3-Chloro-5-((methoxymethoxy)methyl)picolinonitrile was prepared from2,3-dichloro-5-((methoxymethoxy)methyl)pyridine (from the previous step)following the procedures described for Example 42/Step 1.

Step 3: 3-chloro-5-(hydroxymethyl)picolinonitrile

To a stirred solution of 2,3-dichloro-5-((methoxymethoxy)methyl)pyridine(from the previous step) in methanol (0.2 M) was added conc. HCl (10eq). After stirring at room temperature overnight the reaction mixturewas concentrated under vacuum and the resulting crude was purified bychromatography (silica gel, 20-50% EtOAc in hexanes) to afford3-chloro-5-(hydroxymethyl)picolinonitrile.

Step 4: 3-chloro-5-(chloromethyl)picolinonitrile

To a stirred solution of 3-chloro-5-(hydroxymethyl)picolinonitrile (fromthe previous step) in CH₂Cl₂ (0.2 M) at 0° C. was added thionyl chloride(10 eq). After stirring at room temperature overnight the reactionmixture was concentrated under vacuum and the resulting crude waspurified by chromatography (silica gel, 20-50% EtOAc in hexanes) toafford 3-chloro-5-(chloromethyl)picolinonitrile as a colorless oil.

Step 5: 3-chloro-5-(cyanomethyl)picolinonitrile

To a solution of 3-chloro-5-(chloromethyl)picolinonitrile (from theprevious step) in DMSO (0.2 M) was added sodium cyanide (1.25 eq). Thereaction mixture was heated at 130° C. under microwave irradiation. Thereaction mixture taken up in water and EtOAc, and extracted with EtOAc.Organic phases were dried over anhydrous Na₂SO₄, and concentrated. Flashchromatography (silica gel, 20-50% EtOAc in hexanes) of the crudeafforded 3-chloro-5-(cyanomethyl)picolinonitrile.

Step 6: 2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)acetonitrile

2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)acetonitrile wasprepared from 3-chloro-5-(cyanomethyl)picolinonitrile (from the previousstep) following the procedures described for Example 44/Step 4. ¹H NMR(methanol-d₄): δ 8.79 (d, 1H), 8.78 (d, 1H), 8.20 (d, 1H), 7.66 (s, 2H),7.36 (s, 1H), 7.18 (dd, 1H), 4.15 (d, 2H), 2.43 (s, 3H). LRMS[M+H]=249.1

Example 57 N-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)acetamide

Step 1: N-(5,6-dichloropyridin-3-yl)acetamide

To a stirred solution of 5,6-dichloropyridin-3-amine (commerciallyavailable) and triethyl amine (3 eq) in CH₂Cl₂ (0.2 M) at 0° C. wasadded acetyl chloride (2 eq). After stirring at room temperatureovernight the reaction mixture was concentrated under vacuum and theresulting crude residue was purified by chromatography (silica gel,20-50% EtOAc in hexanes) to affordN-(5,6-dichloropyridin-3-yl)acetamide.

Step 2: N-(5-chloro-6-cyanopyridin-3-yl)acetamide

N-(5-chloro-6-cyanopyridin-3-yl)acetamide was prepared fromN-(5,6-dichloropyridin-3-yl)acetamide (from the previous step) followingthe procedures described for Example 42/Step 1.

Step 3: N-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)acetamide

N-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)acetamide was preparedfrom N-(5-chloro-6-cyanopyridin-3-yl)acetamide (from the previous step)following the procedures described for Example 44/Step 4. ¹H NMR(DMSO-d₆): δ 10.99 (s, 1H), 8.18 (d, 1H), 8.95 (d, 1H), 8.12 (d, 1H),7.44 (s, 1H), 7.35 (dd, 1H), 2.43 (s, 3H), 2.16 (s, 3H). LRMS[M+H]=267.1

Example 582-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)-1-(2,4-dimethylphenyl)ethanol

Step 1: 3-bromo-5-(2-(2,4-dimethylphenyl)-2-hydroxyethyl)picolinonitrile

3-bromo-5-(2-(2,4-dimethylphenyl)-2-hydroxyethyl)picolinonitrile wasprepared from 3-bromo-5-methylpicolinonitrile (Example 55/Step 1) and2,4-dimethylbenzaldehyde following the procedures described for Example53/Step 3.

Step 2:2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)-1-(2,4-dimethylphenyl)ethanol

2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)-1-(2,4-dimethylphenyl)ethanolwas prepared from3-bromo-5-(2-(2,4-dimethylphenyl)-2-hydroxyethyl)picolinonitrile (fromthe previous step) following the procedures described for Example53/Step 4. ¹H NMR (CDCl₃): δ 8.67 (d, 1H), 8.45 (d, 1H), 8.06 (d, 1H),7.57 (s, 1H), 7.42 (d, 1H), 7.23 (d, 1H), 7.11 (d, 1H), 7.01 (s, 1H),5.31 (dd, 1H), 3.28-3.25 (m, 2H), 2.53 (s, 3H), 2.35 (s, 3H), 2.33 (s,3H). LRMS [M+H]=358.2

Example 592-(2-(6-methoxy-4-methylpyridin-3-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 2-methoxy-4-methyl-5-((triethylsilyl)ethynyl)pyridine

2-Methoxy-4-methyl-5-((triethylsilyl)ethynyl)pyridine was prepared from5-bromo-2-methoxy-4-methylpyridine (commercially available) followingthe procedures described for Example 44/Step 1.

Step 2: 5-ethynyl-2-methoxy-4-methylpyridine

5-Ethynyl-2-methoxy-4-methylpyridine was prepared from2-methoxy-4-methyl-5-((triethylsilyl)ethynyl)pyridine (from the previousstep) following the procedures described for Example 44/Step 2.

Step 3:3-chloro-5-((6-methoxy-4-methylpyridin-3-yl)ethynyl)picolinonitrile

3-Chloro-5-((6-methoxy-4-methylpyridin-3-yl)ethynyl)picolinonitrile wasprepared from 5-ethynyl-2-methoxy-4-methylpyridine (from the previousstep) following the procedures described for Example 44/Step 3.

Step 4:2-((6-methoxy-4-methylpyridin-3-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-((6-Methoxy-4-methylpyridin-3-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from3-chloro-5-((6-methoxy-4-methylpyridin-3-yl)ethynyl)picolinonitrile(from the previous step) following the procedures described for Example44/Step 4.

Step 5:2-(2-(6-methoxy-4-methylpyridin-3-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(2-(6-Methoxy-4-methylpyridin-3-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from2-((6-methoxy-4-methylpyridin-3-yl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) following the procedures described for Example44/Step 5. ¹H NMR (CDCl₃): δ 8.65 (d, 1H), 8.43 (d, 1H), 8.13 (d, 1H),7.87 (s, 1H), 7.57 (s, 1H), 7.24 (dd, 1H), 6.60 (s, 1H), 6.39 (bs, 2H),3.91 (s, 3H), 3.17-3.11 (dd, 2H), 3.03-2.98 (dd, 2H), 2.54 (s, 3H), 2.28(s, 3H). LRMS [M+H]=359.2

Example 604-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)butan-1-ol

Step 1: 4-(4-((trimethylsilyl)ethynyl)phenyl)but-3-yn-1-ol

4-(4-((trimethylsilyl)ethynyl)phenyl)but-3-yn-1-ol was prepared from((4-bromophenyl)ethynyl)trimethylsilane (commercially available) andbut-3-yn-1-ol (commercially available) following the proceduresdescribed for Example 44/Step 1.

Step 2: 4-(4-ethynylphenyl)but-3-yn-1-ol

4-(4-ethynylphenyl)but-3-yn-1-ol was prepared from4-(4-((trimethylsilyl)ethynyl)phenyl)but-3-yn-1-ol following theprocedures described for Example 44/Step 2.

Step 3:5-((4-(4-hydroxybut-1-ynyl)phenyl)ethynyl)-3-methylpicolinonitrile

5-((4-(4-hydroxybut-1-ynyl)phenyl)ethynyl)-3-methylpicolinonitrile wasprepared from 4-(4-ethynylphenyl)but-3-yn-1-ol (from the previous step)following the procedures described for Example 44/Step 3.

Step 4:4-(4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)but-2-yn-1-ol

4-(4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)but-2-yn-1-olwas prepared from5-((4-(4-hydroxybut-1-ynyl)phenyl)ethynyl)-3-methylpicolinonitrile (fromthe previous step) following the procedures described for Example44/Step 4.

Step 5:4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)butan-1-ol

4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)butan-1-olwas prepared from4-(4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)but-2-yn-1-ol(from the previous step) following the procedures described for Example44/Step 5. ¹H NMR (CDCl₃): δ 8.58 (d, 1H), 8.36 (d, 1H), 8.07 (d, 1H),7.53 (s, 1H), 7.20 (dd, 1H), 7.10 (dd, 4H), 6.20 (bs, 2H), 3.68 (t, 2H),3.20-3.15 (dd, 2H), 3.06-3.01 (dd, 2H), 2.64 (t, 2H), 2.52 (s, 3H),1.75-1.57 (m, 4H). LRMS [M+H]=386.2

Example 61 methyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propanoate

Step 1: methyl 3-(4-iodophenyl)propanoate

To a stirred solution of 3-(4-iodophenyl)propanoic acid (commerciallyavailable) in toluene and methanol (9:1, 0.2 M) 0° C. was added(diazomethyl)trimethylsilane (1 N solution in Et₂O, 2 eq). Afterstirring at room temperature overnight the reaction mixture wasconcentrated under vacuum and the resulting crude residue was purifiedby chromatography (silica gel, 20-50% EtOAc in hexanes) to afford methyl3-(4-iodophenyl)propanoate.

Step 2: methyl 3-(4-ethynylphenyl)propanoate

Methyl 3-(4-ethynylphenyl)propanoate was prepared from methyl3-(4-iodophenyl)propanoate (from the previous step) following theprocedures described for Example 44/Steps 1 and 2.

Step 3: methyl3-(4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)phenyl)propanoate

Methyl 3-(4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)phenyl)propanoate wasprepared from methyl 3-(4-ethynylphenyl)propanoate (from the previousstep) following the procedures described for Example 44/Step 3.

Step 4: methyl3-(4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)propanoate

Methyl3-(4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)propanoatewas prepared from methyl3-(4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)phenyl)propanoate (from theprevious step) following the procedures described for Example 44/Step 4.

Step 5: methyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propanoate

Methyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propanoatewas prepared from methyl3-(4-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)propanoate(from the previous step) following the procedures described for Example44/Step 5. ¹H NMR (DMSO-d₆): δ 8.83 (d, 1H), 8.72 (d, 1H), 8.32 (d, 1H),7.35 (s, 1H), 7.21-7.12 (m, 5H), 7.05 (br s, 2H), 7.05 (dd, 2H), 3.57(s, 3H), 3.19-3.13 (dd, 2H), 3.06-3.00 (dd, 2H), 2.81 (t, 2H), 2.60 (t,2H), 2.45 (s, 3H).

LRMS [M+H]=400.2

Example 623-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propan-1-ol

3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propan-1-olwas prepared from methyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propanoate(from Example 61) following the procedures described for Example 42/Step3. ¹H NMR of the TFA salt: (DMSO-d₆): δ 9.56 (s, 1H), 9.24 (s, 1H), 8.92(d, 1H), 8.81 (d, 1H), 8.43 (d, 1H), 7.44 (d, 1H), 7.35 (d, 1H), 7.13(dd, 2H), 7.05 (dd, 2H), 3.32 (t, 2H), 3.18-3.12 (dd, 2H), 3.02-2.95(dd, 2H), 2.50 (t, 2H), 2.44 (s, 3H), 1.65-1.57 (m, 2H). LRMS[M+H]=372.2

Example 634-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)-2-methylbutan-2-ol

To a solution of methyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propanoate(from Example 61) in THF (0.2 M) at 0° C. was added in a dropwisefashion a solution of methylmagnesium bromide in THF (1.0 M, 2 eq).After stirring at room temperature overnight the reaction mixture wasconcentrated under vacuum and the resulting crude residue was purifiedby chromatography (silica gel, 50-100% EtOAc in hexanes) to afford4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)-2-methylbutan-2-ol.¹H NMR (CDCl₃): δ 8.64 (d, 1H), 8.34 (d, 1H), 8.06 (t, 1H), 7.57 (d,1H), 7.30-7.20 (m, 2H), 7.18-7.07 (m, 4H), 6.67 (bs, 2H), 3.24-3.16 (dd,2H), 3.08-3.01 (dd, 2H), 2.73-2.66 (m, 2H), 2.53 (s, 3H), 1.82-1.75 (m,2H), 1.31 (s, 3H), 1.29 (s, 3H). LRMS [M+H]=400.2

Example 642-(4-(aminomethyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1:4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzonitrile

4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzonitrilewas prepared from 4-ethynylbenzonitrile (commercially available)following the procedures described for Example 44/Steps 3 to 5.

Step 2:2-(4-(aminomethyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzonitrile(from the previous step) in ethanol and ammonium hydroxide (4:1, 0.2 M)stirred at room temperature was added raney nickel (10 eq). The reactionmixture was stirred under hydrogen atmosphere until the conversion wascomplete as shown by TLC. The reaction mixture was filtered through ashort celite pad. The celite pad was washed with EtOAc. Combined organicextracts were concentrated under vacuum and the resulting crude residuewas purified by chromatography (silica gel, 50-100% EtOAc in hexanes) toafford product2-(4-(aminomethyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine.¹H NMR of the TFA salt: (methanol-d₄): δ 8.81 (d, 1H), 8.79 (d, 1H),8.38 (d, 1H), 7.51 (s, 1H), 7.44 (dd, 1H), 7.36 (dd, 4H), 4.07 (s, 2H),3.29 (s, 2H), 3.20-3.14 (dd, 2H), 2.55 (s, 3H). LRMS [M+H]=343.2

Example 65 (E)-ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)acrylate

Step 1:(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)methanol

(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)methanolwas prepared from methyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate(Example 115) following the procedures described for Example 42/Step 3.

Step 2:4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzaldehyde

To a solution of(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)methanol(from the previous step) in DMSO was added 2-iodoxybenzoic acid (IBX,2.5 eq). The reaction was stirred at room temperature for 3 hours beforebeing diluted with water. Extraction with EtOAc followed byconcentration gave a crude residue which was purified by chromatography(silica gel, 50-100% EtOAc in hexanes) to afford4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzaldehyde.

Step 3: (E)-ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)acrylate

To a suspension of NaH (3 eq) in THF (0.2 M) stirred at 0° C. was addedethyl 2-(diethoxyphosphoryl)acetate (commercially available) (3 eq).After stirring for 30 minutes, a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzaldehyde(from the previous step) in THF (0.2 M) was added dropwise. The reactionwas allowed to warm to room temperature and stirred overnight. Thereaction was quenched with sat. NH₄Cl solution, and was extracted withEtOAc. Combined organic extracts were dried and concentrated to give acrude residue which was purified by chromatography (silica gel, 50-100%EtOAc in hexanes) to afford (E)-ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)acrylateas a white solid. ¹H NMR: (CDCl₃): δ 8.54 (d, 1H), 8.29 (d, 1H), 7.99(d, 1H), 7.57 (d, 1H), 7.44 (s, 1H), 7.23 (dd, 1H), 7.11 (dd, 1H), 7.05(d, 1H), 6.33 (d, 1H), 5.93 (s, 2H), 4.19 (q, 2H), 3.10-2.95 (m, 4H),2.44 (s, 3H), 2.23 (s, 3H), 1.26 (t, 3H). LRMS [M+H]=426.2

Example 66 ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propanoate

Ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propanoatewas prepared from (E)-ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)acrylate(from Example 65) following the procedures described for Example 44/Step5. ¹H NMR: (CDCl₃): δ 8.55 (d, 1H), 8.26 (d, 1H), 7.99 (d, 1H), 7.45 (s,1H), 7.12 (dd, 1H), 6.98-6.88 (m, 3H), 6.02 (s, 2H), 4.06 (q, 2H), 3.04(dd, 2H), 2.93 (dd, 2H), 2.83 (t, 2H), 2.53 (t, 2H), 2.44 (s, 3H), 2.19(s, 3H), 1.17 (t, 3H). LRMS [M+H]=428.2

Example 672-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzyl)propane-1,3-diol

Step 1: diethyl2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzyl)malonate

To a stirred solution of(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)methanol(from Example 65/Step 1) (0.2 M) and diethyl malonate (2 eq) in drytoluene was added tributylphosphine (2 eq) andN¹,N¹,N²,N²-tetramethyldiazene-1,2-dicarboxamide (2 eq). The reactionmixture was stirred at 120° C. overnight. Upon completion of thereaction, the reaction mixture was concentrated under vacuum and theresulting crude residue was purified by chromatography (silica gel,50-100% EtOAc in hexanes) to afford diethyl2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzyl)malonateas a white solid.

Step 2:2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzyl)propane-1,3-diol

2-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzyl)propane-1,3-diolwas prepared from diethyl2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzyl)malonate(from the previous step) following the procedures described for Example42/Step 3. ¹H NMR: (methanol-d₄): δ 8.51 (d, 1H), 8.39 (d, 1H), 8.05 (d,1H), 7.45 (s, 1H), 7.10 (dd, 1H), 6.91-6.87 (m, 2H), 6.83 (dd, 1H), 3.42(d, 4H), 3.08-3.02 (m, 2H), 2.96-2.91 (m, 2H), 2.47 (d, 2H), 2.38 (s,3H), 2.13 (s, 3H).

LRMS [M+H]=416.2

Example 683-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propanoicacid

A solution of ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propanoate(from Example 66) in 1 N NaOH, THF and methanol (1:5:2, 0.1 N) washeated at 60° C. for 3 hours. After cooling to room temperature thereaction mixture was neutralized with 1 N HCl to pH7, and wasconcentrated to give a crude residue which was purified bychromatography (silica gel, 0-20% methanol in dichloromethane) to afford(E)-ethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)acrylateas a white solid. ¹H NMR: (methanol-d₄): δ 8.73 (d, 1H), 8.54 (d, 1H),8.20 (d, 1H), 7.45 (s, 1H), 7.37 (d, 1H), 7.00-6.97 (m, 2H), 6.92 (d,1H), 3.19 (t, 2H), 3.04 (t, 2H), 2.81 (t, 2H), 2.53 (t, 2H), 2.50 (s,3H), 2.25 (s, 3H). LRMS [M+H]=400.2

Example 695-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridine-8-carbaldehyde

5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridine-8-carbaldehydewas prepared from(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol(from Example 108) following the procedures described for Example65/Step 2. ¹H NMR: (CDCl₃): δ 10.19 (s, 1H), 8.74 (d, 1H), 8.43 (d, 1H),8.32 (d, 1H), 8.18 (d, 1H), 7.88 (dd, 1H), 7.00 (d, 1H), 6.76 (d, 1H),6.70 (dd, 1H), 6.30 (s, 2H), 3.80 (s, 3H), 3.16 (dd, 2H), 3.02 (dd, 2H),2.29 (s, 3H). LRMS [M+H]=372.2

Example 70 ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoate

Step 1: ethyl 4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)benzoate

A solution of 3,5-dichloropicolinonitrile (commercially available) (1.0eq.), ethyl 4-ethynylbenzoate (commercially available) (1.0 eq.),trans-dichlororbis(triphenylphosphine)palladium (II) (10 mol %), copper(I) iodide (20 mol %), and triethylamine (5.0 eq.) in DMF (0.3 M) wasstirred at 50° C. for 3 hours. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and 10% aqueous ammoniumhydroxide. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH®, system (ISCO) using 0-20% ethyl acetate in hexane to giveethyl 4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)benzoate as a whitesolid.

Step 2: ethyl4-O-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)benzoate

A solution of ethyl 4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)benzoate(from the previous step) (1.0 eq.), tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (2.6 eq.), tetrakis(triphenylphosphine)palladium(10 mol %), and potassium carbonate (5.3 eq.) in toluene/ethanol (2:1,0.2 M) was stirred at 100° C. overnight. After cooling to ambienttemperature, the reaction mixture was diluted with 2% MeOH in DCM. Thetwo phases were separated. The combined organic layers were washed withbrine, dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-40% ethyl acetate in toluene to give ethyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)benzoate.

Step 3: ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoate

A solution of ethyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)benzoate(from the previous step) (1.0 eq.) in THF/ethyl acetate (1:1, 0.05M) wasflushed with nitrogen and palladium on carbon (10 wt %) was added. Thereaction vessel was evacuated, flushed with hydrogen, and stirredovernight at room temperature. The reaction mixture was filtered throughcelite, washed with 2% MeOH in DCM, and concentrated en vaccuo. Thecrude material was purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-5% MeOH in DCM to give ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoate. ¹HNMR (Acetone-d₆): δ 8.80 (s, 1H), 8.69 (s, 1H), 8.25 (d, 1H), 7.90 (d,2H), 7.40-7.42 (m, 3H), 7.12 (d, 1H), 6.55 (br, 2H), 4.28 (q, 2H),3.2-3.3 (m, 4H), 2.44 (s, 3H), 1.31 (t, 3H). LRMS [M+H]=386.2

Example 718-methyl-2-(4-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-(p-tolylethynyl)picolinonitrile

A solution of 3,5-dichloropicolinonitrile (commercially available) (1.0eq.), 1-ethynyl-4-methylbenzene (commercially available) (1.0 eq.),trans-dichlororbis(triphenylphosphine)palladium (II) (10 mol %), copper(I) iodide (20 mol %), and triethylamine (5.0 eq.) in DMF (0.3 M) wasstirred at 50° C. for 3 hours. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and 10% aqueous ammoniumhydroxide. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by stirring in hot ether/hexanemixtures and filtered to give3-chloro-5-(p-tolylethynyl)picolinonitrile.

Step 2: 8-methyl-2-(p-tolylethynyl)benzo[f][1,7]naphthyridin-5-amine

A solution of 3-chloro-5-(p-tolylethynyl)picolinonitrile (from theprevious step) (1.0 eq.), tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.2 eq.), tetrakis(triphenylphosphine)palladium(10 mol %), and 2N sodium carbonate aqueous solution (4.0 eq.) intoluene/ethanol (2:1, 0.2 M) was stirred at 100° C. overnight. Aftercooling to ambient temperature, the reaction mixture was diluted with 2%MeOH in DCM. The two phases were separated, and the aqueous layer wasextracted with 2% MeOH in DCM twice. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-40% ethyl acetate in toluene to give8-methyl-2-(p-tolylethynyl)benzo[f][1,7]naphthyridin-5-amine.

Step 3: 8-methyl-2-(4-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine

A solution of8-methyl-2-(p-tolylethynyl)benzo[f][1,7]naphthyridin-5-amine (from theprevious step) (1.0 eq.) in EtOH/ethyl acetate (1:1, 0.05M) was flushedwith nitrogen and palladium on carbon (10 wt %) was added. The reactionvessel was evacuated, flushed with hydrogen, and stirred overnight atroom temperature. The reaction mixture was filtered through celite,washed with 2% MeOH in DCM, and concentrated en vaccuo. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-5% MeOH in DCM to give8-methyl-2-(4-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine. ¹H NMR(Acetone-d₆): δ 8.74 (s, 1H), 8.68 (s, 1H), 8.24 (d, 1H), 7.41 (s, 1H),7.13-7.15 (m, 3H), 7.06 (d, 2H), 6.6 (br, 2H), 3.19 (t, 2H), 3.06 (t,2H), 2.44 (s, 3H), 2.25 (s, 3H). LRMS [M+H]=328.1

Example 722-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propan-2-ol

To a solution of ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoate(from Example 70) (1.0 eq.) in DCM at 0° C. was added 3.0 M methylmagnesium iodide (10 eq.) in ether and warmed to room temperatureovernight. The reaction was cooled to 0° C. and quenched with 1N HClaqueous solution and ether. After stirring for 15 minutes, the reactionmixture was neutralized with saturated aqueous sodium bicarbonatesolution. The two phases were separated, and the aqueous layer wasextracted with ether. The combined organic layers were washed withbrine, dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by RP-HPLC using a 10-50% MeCN in water gradientfollowed by extraction in DCM to give2-(4-(2-(5-amino-8methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propan-2-ol.¹H NMR (Acetone-d₆): δ 8.73 (m, 2H), 8.22 (d, 1H), 7.40-7.44 (m, 3H),7.20 (d, 2H), 7.12 (d, 1H), 6.5 (br, 2H), 3.94 (s, 1H), 3.21 (t, 2H),3.08 (t, 2H), 2.44 (s, 3H), 1.47 (s, 6H). LRMS [M+H]=372.2

Example 73(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)methanol

To a solution of ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoate(Example 70) (1.0 eq.) in THF (0.1 M) at 0° C. was added 1.0 M lithiumtriethylborohydride in THF (10 eq.) and warmed to room temperature over2 hours. 1N HCl aqueous solution was added slowly to quench thereaction, and the mixture was heated to reflux for 30 minutes. Thereaction mixture was neutralized with saturated aqueous sodiumbicarbonate solution. The two phases were separated, and the aqueouslayer was extracted with ethyl acetate (EA). The combined organic layerswere washed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by RP-HPLC using a 10-50% MeCNin water gradient followed by extraction in DCM to give(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)methanol.¹H NMR (Acetone-d₆): δ 8.77 (s, 1H), 8.69 (s, 1H), 8.26 (d, 1H), 7.40(s, 1H), 7.21-7.28 (m, 4H), 7.13 (d, 1H), 6.5 (br, 2H), 4.56 (s, 2H),4.1 (br t, 1H), 3.10-3.23 (m, 4H), 2.44 (s, 3H). LRMS [M+H]=344.2

Example 74 ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate

Step 1: ethyl 4-bromo-3-methylbenzoate

To a solution of 4-bromo-3-methylbenzoic acid (commercially available)(1.0 eq.) in EtOH (0.3 M) was added thionyl chloride (1.5 eq.) andheated to reflux for 2 hours. The solvent was concentrated en vaccuo,and the residue was diluted in ether and neutralized with saturatedaqueous sodium bicarbonate solution. The two phases were separated, andthe aqueous layer was extracted with ether. The combined organic layerswere washed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo to give ethyl 4-bromo-3-methylbenzoate.

Step 2: ethyl 3-methyl-4-((triethylsilyl)ethynyl)benzoate

A solution of ethyl 4-bromo-3-methylbenzoate (from the previous step)(1.0 eq.), triethyl(ethynyl)silane (1.1 eq.),trans-dichlororbis(triphenylphosphine)palladium (II) (10 mol %), copper(I) iodide (20 mol %), and triethylamine (5.0 eq.) in DMF (0.3 M) wasstirred at 60° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and 10% aqueous ammoniumhydroxide. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-5% ethyl acetate in hexane to giveethyl 3-methyl-4-((triethylsilyl)ethynyl)benzoate as a yellow oil.

Step 3: ethyl 4-ethynyl-3-methylbenzoate

To a solution of ethyl 3-methyl-4-((triethylsilyl)ethynyl)benzoate (fromthe previous step) (1.0 eq.) in THF (0.3 M) at 0° C. was added dropwise1.0 M TBAF in THF (1.2 eq.). After stirring for 10 minutes at 0° C., thereaction was quenched with saturated aqueous sodium bicarbonatesolution. The two phases were separated, and the aqueous layer wasextracted with ether. The combined organic layers were washed withbrine, dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-5% ethyl acetate in hexane to give ethyl4-ethynyl-3-methylbenzoate as a white solid.

Step 4: ethyl 4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)-3-methylbenzoate

A solution of 3,5-dichloropicolinonitrile (1.0 eq.), ethyl4-ethynyl-3-methylbenzoate (from the previous step) (1.0 eq.),trans-dichlororbis(triphenylphosphine)palladium (II) (10 mol %), copper(1) iodide (20 mol %), and triethylamine (5.0 eq.) in DMF (0.3 M) wasstirred at 50° C. for 3 hours. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and 10% aqueous ammoniumhydroxide. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ethyl acetate in hexane to giveethyl 4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)-3-methylbenzoate as awhite solid.

Step 5: ethyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)-3-methylbenzoate

A solution of ethyl4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)-3-methylbenzoate (from theprevious step) (1.0 eq.), tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.1 eq.), tetrakis(triphenylphosphine)palladium(8 mol %), and potassium carbonate (3.0 eq.) in toluene/ethanol (9:1,0.2 M) was stirred at 100° C. overnight. After cooling to ambienttemperature, the reaction mixture was diluted with 2% MeOH in DCM. Thetwo phases were separated. The combined organic layers were washed withbrine, dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-40% ethyl acetate in toluene to give ethyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)-3-methylbenzoate.

Step 6: ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate

A solution of ethyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)-3-methylbenzoate(from the previous step) (1.0 eq.) in THF/ethyl acetate (1:1, 0.05M) wasflushed with nitrogen and added 10% palladium on carbon (10 wt %). Thereaction vessel was evacuated, flushed with hydrogen, and stirredovernight at room temperature. The reaction mixture was filtered throughcelite, washed with 2% MeOH in DCM, and concentrated en vaccuo. Thecrude material was purified by flash chromatography on a COMBIFLASH®system (ISCO) using 30-100% EA in hexane to give ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate.¹H NMR (Acetone-d₆): δ 8.79 (s, 1H), 8.71 (s, 1H), 8.24 (d, 1H), 7.80(s, 1H), 7.73 (d, 1H), 7.40 (s, 1H), 7.31 (d, 1H), 7.12 (d, 1H), 6.5(br, 2H), 4.29 (q, 2H), 3.19-3.22 (m, 4H), 2.44 (s, 3H), 2.39 (s, 3H),1.31 (t, 3H).

LRMS [M+H]=400.2

Example 754-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoicacid

To a solution of ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate(from Example 74) (1.0 eq.) in EtOH was added 1N aqueous sodiumhydroxide (1.5 eq.) and heated to 80° C. for 5 hours. The reactionmixture was neutralized by adding 1N aqueous HCl (1.5 eq.) andconcentrated en vaccuo. The crude material was purified by RP-HPLC usinga 10-50% MeCN in water gradient followed by concentration en vaccuo togive the TFA salt. ¹H NMR (DMSO-d₆) of the TFA salt: δ 7.94-7.96 (m,2H), 7.55 (d, 1H), 7.00 (s, 1H), 6.91 (d, 1H), 6.62-6.66 (m, 2H), 6.39(d, 1H), 2.36-2.5 (m, 4H), 1.73 (s, 3H), 1.54 (s, 3H). LRMS [M+H]=372.2

Example 76(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)methanol

To a solution of ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate(from Example 74) (1.0 eq.) in THF (0.1M) at −78° C. was added 1.0 MDIBAL-H in toluene (10 eq.) and warmed to room temperature over 2 hours.1.5 M Rochelle salt aqueous solution was added slowly to quench thereaction followed by addition of EA, and the mixture was stirred for 45minutes. The two phases were separated, and the aqueous layer wasextracted twice with EA. The combined organic layers were washed withbrine, dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by RP-HPLC using a 10-50% MeCN in water gradientfollowed by extraction in DCM to give(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)methanol.¹H NMR (Acetone-d₆): δ 8.77 (s, 1H), 8.71 (s, 1H), 8.25 (d, 1H), 7.41(s, 1H), 7.10-7.15 (m, 4H), 6.5 (br, 2H), 4.54 (s, 2H), 4.05 (br, 1H),3.08-3.18 (m, 4H), 2.44 (s, 3H), 2.31 (s, 3H). LRMS [M+H]=358.2

Example 778-methyl-2-(2,4,6-trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-(mesitylethynyl)picolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),2-ethynyl-1,3,5-trimethylbenzene (commercially available) (1.0 eq.),trans-dichlororbis(triphenylphosphine)palladium (II) (10 mol %), copper(I) iodide (20 mol %), and triethylamine (5.0 eq.) in DMF (0.3 M) wasstirred at 50° C. for 3 hours. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and 10% aqueous ammoniumhydroxide. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ethyl acetate in hexane to give3-chloro-5-(mesitylethynyl)picolinonitrile a as white solid.

Step 2: 2-(mesitylethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of 3-chloro-5-(mesitylethynyl)picolinonitrile (from theprevious step) (1.0 eq.), tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.1 eq.), tetrakis(triphenylphosphine)palladium(8 mol %), and 2N aqueous sodium carbonate solution (3.0 eq.) intoluene/ethanol (4:1, 0.2 M) was stirred at 100° C. overnight. Aftercooling to ambient temperature, the reaction mixture was diluted with 2%MeOH in DCM. The two phases were separated. The combined organic layerswere washed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-40% ethyl acetate in toluene to give2-(mesitylethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine.

Step 3:8-methyl-2-(2,4,6-trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine

A solution of2-(mesitylethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine (from theprevious step) (1.0 eq.) in EtOH (0.05M) was flushed with nitrogen andadded palladium on carbon (10 wt %). The reaction vessel was evacuated,flushed with hydrogen, and stirred overnight at rt. The reaction mixturewas filtered through celite, washed with 2% MeOH in DCM, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-50% ethyl acetatein hexane to give8-methyl-2-(2,4,6-trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine.¹H NMR (Acetone-d₆): δ 8.73-8.74 (m, 2H), 8.25 (d, 1H), 7.42 (s, 1H),7.14 (d, 1H), 6.83 (s, 2H), 6.55 (br, 2H), 3.07 (m, 4H), 2.47 (s, 3H),2.29 (s, 6H), 2.22 (s, 3H). LRMS [M+H]=356.2

Example 782-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol

To a solution of ethyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate(from Example 74) (1.0 eq.) in DCM at 0° C. was added 3.0 M methylmagnesium iodide (10 eq.) in ether and warmed to room temperatureovernight. The reaction was cooled to 0° C. and quenched with water.After stirring for 15 min, the reaction mixture was neutralized withsaturated aqueous sodium bicarbonate solution and added EA. The twophases were separated, and the aqueous layer was extracted three timeswith EA. The combined organic layers were washed with brine, dried overanhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-5% MeOH in DCM to give a2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol.¹H NMR (Acetone-d₆): δ 8.72-8.75 (m, 2H), 8.23 (d, 1H), 7.41 (s, 1H),7.32 (s, 1H), 7.25 (d, 1H), 7.12-7.14 (m, 2H), 6.6 (br, 2H), 3.91 (s,1H), 3.07-3.18 (m, 4H), 2.44 (s, 3H), 2.31 (s, 3H), 1.48 (s, 6H). LRMS[M+H]=386.2

Example 798-methyl-2-(4-propoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-5-((4-propoxyphenyl)ethynyl)picolinonitrile

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),1-ethynyl-4-propoxybenzene (commercially available) (1.0 eq.),trans-dichlororbis(triphenylphosphine)palladium (II) (10 mol %), copper(I) iodide (20 mol %), and triethylamine (5.0 eq.) in DMF (0.3 M) wasstirred at 50° C. for 3 hours. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and 10% aqueous ammoniumhydroxide. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-10% ethyl acetate in hexane to give3-chloro-5-((4-propoxyphenyl)ethynyl)picolinonitrile as a white solid.

Step 2: 3-chloro-5-(4-propoxyphenethyl)picolinonitrile

A solution of 3-chloro-5-((4-propoxyphenyl)ethynyl)picolinonitrile (fromthe previous step) (1.0 eq.) in EtOH (0.05M) was flushed with nitrogenand added platinum (VI) oxide (0.5 eq.). The reaction vessel wasevacuated, flushed with hydrogen, and stirred for 5 hours at roomtemperature. The reaction mixture was filtered through celite, washedwith 2% MeOH in DCM, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-15% ethyl acetate in hexane to give3-chloro-5-(4-propoxyphenethyl)picolinonitrile.

Step 3: 8-methyl-2-(4-propoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine

A solution of 3-chloro-5-(4-propoxyphenethyl)picolinonitrile (from theprevious step) (1.0 eq.), tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.1 eq.), tetrakis(triphenylphosphine)palladium(8 mol %), and 2N aqueous sodium carbonate solution (3.0 eq.) in toluene(0.2 M) was stirred at 100° C. overnight. After cooling to ambienttemperature, the reaction mixture was diluted with 2% MeOH in DCM. Thetwo phases were separated. The combined organic layers were washed withbrine, dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-40% ethyl acetate in toluene to give8-methyl-2-(4-propoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine. ¹H NMR(Acetone-d₆): δ 8.74 (s, 1H), 8.67 (s, 1H), 8.24 (d, 1H), 7.41 (s, 1H),7.15-7.17 (m, 3H), 6.81 (d, 2H), 6.5 (br, 2H), 3.87 (t, 2H), 3.18 (t,2H), 3.04 (t, 2H), 2.44 (s, 3H), 1.73 (m, 2H), 0.99 (t, 3H). LRMS[M+H]=372.2

Example 80 (E)-ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)acrylate

(E)-ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)acrylatewas prepared from5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridine-8-carbaldehyde(from Example 69) and ethyl 2-(diethoxyphosphoryl)acetate (commerciallyavailable) following the procedures described for Example 65/Step 3.LRMS [M+H]=442.2

Example 81(E)-3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)acrylicacid

(E)-3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)acrylicacid was prepared from (E)-ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)acrylate(from Example 80) following the procedures described for Example 68. ¹HNMR of TFA salt (DMSO-d₆): δ 12.66 (s, 1H), 9.09 (s, 1H), 8.88 (s, 1H),8.66 (d, 1H), 7.95 (d, 1H), 7.91 (s, 1H), 7.75 (d, 1H), 7.10 (d, 1H),6.77-6.71 (m, 2H), 6.68 (dd, 1H), 3.70 (s, 3H), 3.16 (t, 2H), 3.00 (t,2H), 2.30 (s, 3H). LRMS [M+H]=414.2

Example 82 ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoate

Ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoatewas prepared from (E)-ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)acrylate(from Example 80) following the procedures described for Example 44/Step5. ¹H NMR (CDCl₃): δ 8.63 (d, 1H), 8.37 (d, 1H), 8.13 (d, 1H), 7.56 (d,1H), 7.24 (dd, 1H), 7.02 (d, 1H), 6.75 (d, 1H), 6.69 (dd, 1H), 6.15 (brs, 2H), 4.17 (q, 2H), 3.79 (s, 3H), 3.12 (dd, 4H), 2.99 (dd, 2H), 2.75(t, 2H), 2.29 (s, 3H), 1.27 (t, 2H), 0.99 (t, 3H). LRMS [M+H]=444.2

Example 833-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoicacid

3-(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoicacid was prepared from ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoate(from Example 82) following the procedures described for Example 68. ¹HNMR (DMSO-d₆): δ 12.18 (s, 1H), 8.84 (d, 1H), 8.70 (d, 1H), 8.36 (d,1H), 7.39 (d, 1H), 7.20 (dd, 1H), 7.09 (m, 2H), 6.74 (d, 1H), 6.68 (dd,1H), 3.70 (s, 3H), 3.09 (dd, 2H), 2.96 (dd, 4H), 2.63 (t, 2H), 2.27 (s,3H). LRMS [M+H]=416.2

Example 843-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propan-1-ol

3-(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propan-1-olwas prepared from ethyl3-(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoate(from Example 82) following the procedures described for Example 42/Step3. ¹H NMR (CDCl₃): δ 8.54 (d, 1H), 8.30 (d, 1H), 8.05 (d, 1H), 7.48 (d,1H), 7.15 (dd, 1H), 6.93 (d, 1H), 6.66 (d, 1H), 6.61 (dd, 1H), 5.98 (brs, 2H), 3.71 (s, 3H), 3.66 (t, 2H), 3.03 (dd, 2H), 2.91 (dd, 2H), 2.81(t, 2H), 2.20 (s, 3H), 1.98-1.90 (m, 2H). LRMS [M+H]=402.2

Example 85 (5-aminobenzo[f][1,7]naphthyridin-8-yl)methanol

Step 1: 2-(tert-butoxycarbonylamino)-4-(methoxycarbonyl)phenylboronicacid

A solution of 2-amino-4-(methoxycarbonyl)phenylboronic acidhydrochloride (commercially available) (1.0 eq.), triethylamine (3.0eq.), di-tert-butyl dicarbonate (1.1 eq.), and DMAP (0.1 eq.) in CH₃CN(0.3 M) was stirred at 40° C. overnight. After cooling to ambienttemperature, the reaction mixture was concentrated en vaccuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-30% MeOH/DCM to give2-(tert-butoxycarbonylamino)-4-(methoxycarbonyl)phenylboronic acid as abrown solid.

Step 2: methyl 5-aminobenzo[f][1,7]naphthyridine-8-carboxylate

A solution of2-(tert-butoxycarbonylamino)-4-(methoxycarbonyl)phenylboronic acid (fromthe previous step) (1.0 eq.) and 3-bromopicolinonitrile (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was filtered to collect the precipitate. Theprecipitate was rinsed with EtOAc to give methyl5-aminobenzo[f][1,7]naphthyridine-8-carboxylate as a pale brown solid.

Step 3: (5-aminobenzo[f][1,7]naphthyridin-8-yl)methanol

To a solution of methyl 5-aminobenzo[f][1,7]naphthyridine-8-carboxylate(from the previous step) (1.0 eq.) in EtOH (0.03M) was added NaBH₄ (10eq.) at 25° C. The solution was heated to 80° C. for 5 hours. Aftercooling to ambient temperature, the reaction mixture was concentrated envaccuo. The residue was portionized between saturated NaHCO₃ and EtOAc.The layers were separated and aqueous layer was extracted with EtOActwice. The combined organic layer was washed with brine, dried overMgSO₄ and concentrated en vaccuo to obtain a crude residue. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-10% MeOH/DCM to give(5-aminobenzo[f][1,7]naphthyridin-8-yl)methanol as an off white solid:¹H NMR (methanol-d₄): δ 8.82 (dd, 1H), 8.77 (dd, 1H), 7.26 (d, 1H), 7.70(dd, 1H), 7.50 (d, 1H), 7.27 (dd, 1H), 4.66 (s, 2H). LRMS [M+H]=226.1.

Example 86 5-aminobenzo[f][1,7]naphthyridin-8-ol

To a solution of 8-methoxybenzo[f][1,7]naphthyridin-5-amine (fromExample 10) (1.0 eq.) in DCM (0.04 M) was added BBr₃ (2.5 eq.) dropwiseunder N₂ at −20° C. The reaction was allowed to warm to ambienttemperature over 30 minutes. The reaction was then stirred overnight.The reaction was quenched with saturated NaHCO₃ and extracted withEtOAc. The combined organic layer was washed with brine, dried overMgSO₄ and concentrated en vaccuo to obtain a crude residue. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-20% MeOH/DCM to give5-aminobenzo[f][1,7]naphthyridin-8-ol as a yellow solid: ¹H NMR(acetone-d₆): δ 8.90 (dd, 1H), 8.83 (dd, 1H), 8.32 (d, 1H), 7.83 (dd,1H), 7.11 (br s, 2H), 7.10 (d, 1H), 6.96 (dd, 1H), 5.86 (br s, 1H). LRMS[M+H]=212.1.

Example 87 5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde

A solution of (5-aminobenzo[f][1,7]naphthyridin-8-yl)methanol (fromExample 85) (1.0 eq.) and activated MnO₂ (20 eq.) in DCM (0.1 M) wasstirred at ambient temperature over night. The reaction mixture wasdiluted with DCM. The MnO₂ was filtered off, and the filtrate wasconcentrated en vaccuo to obtain a crude residue. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-10% MeOH/DCM to give 5-aminobenzo[f][1,7]naphthyridine-8-carbaldehydeas a yellow solid: ¹H NMR (acetone-d₆): δ 10.19 (s, 1H), 9.14 (dd, 1H),9.01 (dd, 1H), 8.63 (d, 1H), 8.14 (d, 1H), 7.93 (dd, 1H), 7.81 (dd, 1H),6.96 (br s, 2H). LRMS [M+H]=224.1

Example 88 1-(5-aminobenzo[f][1,7]naphthyridin-8-yl)ethanol

To a solution of 5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde (fromExample 87) (1.0 eq.) in THF (0.02M) was added MeLi (2.5 eq.) at −78° C.The reaction was allowed to warm to ambient temperature overnight. Thereaction was quenched by saturated NH₄Cl and extracted with EtOAc. Thecombined organic layer was washed with brine, dried over MgSO₄ andconcentrated en vaccuo to obtain a crude residue. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-5% MeOH/DCM to give 1-(5-aminobenzo[f][1,7]naphthyridin-8-yl)ethanolas a yellow solid: ¹H NMR (methanol-d₄): δ 8.94 (dd, 1H), 8.88 (dd, 1H),8.38 (d, 1H), 7.81 (dd, 1H), 7.62 (d, 1H), 7.41 (dd, 1H), 4.97 (q, 1H),1.53 (d, 3H). LRMS [M+H]=240.1.

Example 89 1-(5-aminobenzo[f][1,7]naphthyridin-8-yl)ethanone

A solution 1-(5-aminobenzo[f][1,7]naphthyridin-8-yl)ethanol (fromExample 88) (1.0 eq.) and activated MnO₂ (20 eq.) in DCM (0.1 M) wasstirred at ambient temperature over night. The reaction mixture wasdiluted with DCM. The MnO₂ was filtered off, and the filtrate wasconcentrated en vaccuo to obtain a crude residue. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-5% MeOH/DCM to give 1-(5-aminobenzo[f][1,7]naphthyridin-8-yl)ethanoneas a yellow solid: ¹H NMR (acetone-d₆): δ 9.11 (dd, 1H), 8.99 (dd, 1H),8.56 (d, 1H), 8.20 (d, 1H), 7.94-7.88 (m, 2H), 6.90 (br s, 2H), 2.70 (s,3H). LRMS [M+H]=238.1.

Example 90 8-isopropylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 2-(5-aminobenzo[f][1,7]naphthyridin-8-yl)propan-2-ol

To a solution of methyl 5-aminobenzo[f][1,7]naphthyridine-8-carboxylate(from Example 85/Step 2) (1.0 eq.) in THF (0.02M) was added MeLi (10eq.) at −78° C. The reaction was allowed to warm to ambient temperatureovernight. The reaction was quenched by saturated NH₄Cl and extractedwith EtOAc. The combined organic layer was washed with brine, dried overMgSO₄ and concentrated en vaccuo to obtain a crude residue. The crudematerial was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-10% MeOH/DCM to give2-(5-aminobenzo[f][1,7]naphthyridin-8-yl)propan-2-ol as a yellow oil.

Step 2: 8-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine

A solution of 2-(5-aminobenzo[f][1,7]naphthyridin-8-yl)propan-2-ol (fromthe previous step) (1.0 eq.) and p-TsOH (2 eq.) in toluene (0.01 M) wasstirred at 90° C. for 6 hours. The reaction was quenched by saturatedNaHCO₃ and extracted with EtOAc. The combined organic layer was washedwith brine, dried over MgSO₄ and concentrated en vaccuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-5% MeOH/DCM to give8-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine as a yellow solid.

Step 3: 8-isopropylbenzo[f][1,7]naphthyridin-5-amine

A mixture of 8-(prop-1-en-2-yl)benzo[f][1,7]naphthyridin-5-amine (fromthe previous step) (1.0 eq) and Pd/C (wet, 10% wt) in EtOH was stirredunder H₂ balloon overnight. The reaction mixture was diluted with DCM.The Pd/C was filtered off through celite, and the filtrate wasconcentrated en vaccuo to obtain a crude residue. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-60% EtOAc/Hexanes to give 8-isopropylbenzo[f][1,7]naphthyridin-5-amineas a yellow solid: ¹H NMR (acetone-d₆): δ 8.98 (dd, 1H), 8.88 (dd, 1H),8.37 (d, 1H), 7.83 (dd, 1H), 7.49 (d, 1H), 7.27 (dd, 1H), 6.66 (br s,2H), 3.10-3.00 (m, 1H), 1.33 (d, 6H). LRMS [M+11]=238.1.

Example 91 8-vinylbenzo[f][1,7]naphthyridin-5-amine

To a solution of methyl triphenyl phosphonium iodide (6.0 eq.) was addednBuLi (7.0 eq.) at −78° C. The reaction mixture was allowed to warm to0° C. and stirred for 30 minutes (deep orange color). The reaction wasagain cooled down to −78° C. and5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde (from Example 87) (1.0eq.) in THF was introduced dropwised to the reaction. The reaction wasallowed to warm to ambient temperature overnight. The reaction wasquenched by saturated NH₄Cl and extracted with EtOAc. The combinedorganic layer was washed with brine, dried over MgSO₄ and concentrateden vaccuo to obtain a crude residue. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-50%EtOAc/Hexanes to give 8-vinylbenzo[f][1,7]naphthyridin-5-amine as awhite solid: ¹H NMR (acetone-d₆): ¹H NMR (acetone-d₆): δ 9.00 (dd, 1H),8.90 (dd, 1H), 8.41 (d, 1H), 7.84 (dd, 1H), 7.65 (d, 1H), 7.52 (dd, 1H),6.91 (dd, 1H), 6.77 (br s, 2H), 5.97 (dd, 1H), 5.34 (dd, 1H). LRMS[M+H]=222.1.

Example 92 8-ethylbenzo[f][1,7]naphthyridin-5-amine

A mixture of 8-vinylbenzo[f][1,7]naphthyridin-5-amine (1.0 eq) (fromExample 91) and Pd/C (wet, 10% wt) in EtOH was stirred under H₂ balloonovernight. The reaction mixture was diluted with DCM. The Pd/C wasfiltered off through celite, and the filtrate was concentrated en vaccuoto obtain a crude residue. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-60% EtOAc/Hexanesto give 8-ethylbenzo[f][1,7]naphthyridin-5-amine as a white foam: ¹H NMR(acetone-d₆): δ 8.98 (dd, 1H), 8.88 (dd, 1H), 8.35 (d, 1H), 7.82 (dd,1H), 7.46 (d, 1H), 7.22 (dd, 1H), 6.63 (br s, 2H), 2.78 (q, 2H), 1.30(t, 3H). LRMS [M+H]=224.1.

Example 93 8-(methoxymethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 2-chloro-5-(methoxymethyl)phenylcarbamate

To a solution of 2-chloro-5-(methoxymethyl)aniline (commerciallyavailable) (1.0 eq.) in THF (0.2M) at 0° C. under N₂ atmosphere wasadded dropwise 1M NaHMDS (2.5 eq.). The reaction was stirred for 15minutes at 0° C., and a solution of di-tert-butyl dicarbonate in THF wasadded. The reaction was warmed to ambient temperature overnight. Thesolvent was evaporated, and the resulting residue was quenched with 0.1NHCl aqueous solution. The aqueous suspension was extracted twice withEtOAc. The combined organic layers were washed with brine, dried overanhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-30% EtOAc/Hexanes to give tert-butyl2-chloro-5-(methoxymethyl)phenylcarbamate as a colorless oil.

Step 2: tert-butyl5-(methoxymethyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

Tert-butyl 2-chloro-5-(methoxymethyl)phenylcarbamate (from the previousstep) (1.0 eq.),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (3.0 eq.),Pd₂ dba₃ (2.5%), XPhos (10%), and KOAc (3 eq.) were mixed in dioxane(0.2 M) under N₂ atmosphere. The reaction was heated to 110° C. andstirred overnight. The resulting suspension was cooled to ambienttemperature, diluted with ether, filtered through celite, and thefiltrate was concentrated en vaccuo. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-20% EtOAc/Hexanes to give tert-butyl5-(methoxymethyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamateas a white foam.

Step 3: 8-(methoxymethyl)benzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-(methoxymethyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from the previous step) (1.0 eq.) and 3-bromopicolino-nitrile (1.0 eq.)in toluene (0.44 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and 2N aqueouspotassium carbonate solution (2.0 eq.). The reaction was heated to 100°C. and stirred overnight. After cooling to ambient temperature, thereaction mixture was diluted with 2% MeOH in DCM and water. The twophases were separated, and the aqueous layer was extracted twice with 2%MeOH in DCM. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-100% EtOAc/Hexanes to give8-(methoxymethyl)benzo[f][1,7]naphthyridin-5-amine as a white solid: ¹HNMR (methanol-d₄): δ 8.97 (dd, 1H), 8.91 (dd, 1H), 8.41 (dd, 1H), 7.83(dd, 1H), 7.59 (d, 1H), 7.37 (dd, 1H), 4.62 (s, 2H), 3.45 (s, 3H). LRMS[M+H]=240.1.

Example 94 (5-amino-2-phenethylbenzo[f][1,7]naphthyridin-8-yl)methanol

Step 1: methyl5-amino-2-phenethylbenzo[f][1,7]naphthyridine-8-carboxylate

A solution of2-(tert-butoxycarbonylamino)-4-(methoxycarbonyl)phenylboronic acid (fromExample 85/Step 1) (1.0 eq.) and 2-chloro-6-phenethylnicotinonitrile(prepared from (E)-3-chloro-5-styrylpicolinonitrile (from Example32/Step 1) following the procedure described in Example 114/Step 3) (1.0eq.), tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueoussodium carbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with EtOAc and water. The two phases wereseparated, and the aqueous layer was extracted twice with EtOAc. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vaccuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-60%EtOAc/Hexanes to give methyl5-amino-2-phenethylbenzo[f][1,7]naphthyridine-8-carboxylate as a whitesolid.

Step 2: (5-amino-2-phenethylbenzo[f][1,7]naphthyridin-8-yl)methanol

To a solution of methyl5-amino-2-phenethylbenzo[f][1,7]naphthyridine-8-carboxylate (from theprevious step) (1.0 eq.) in THF (0.03M) was added Super-H (10 eq.) at 0°C. The solution was allowed to warm to ambient temperature over 30 min.The reaction was quenched by water until no bubbling. The layers wereseparated and aqueous layer was extracted with EtOAc. The combinedorganic layer was washed with brine, dried over MgSO₄ and concentrateden vaccuo to obtain a crude residue. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-10% MeOH/DCMto give (5-amino-2-phenethylbenzo[f][1,7]naphthyridin-8-yl)methanol asan off white solid: ¹H NMR (methanol-d₄): δ 8.63 (dd, 1H), 8.56 (dd,1H), 8.24 (d, 1H), 7.57 (d, 1H), 7.35 (dd, 1H), 7.27-7.15 (m, 5H), 4.75(s, 2H), 3.20 (t, 2H), 3.06 (t, 2H). LRMS [M+H]=330.1.

Example 95(5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

Step 1: methyl5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridine-8-carboxylate

A solution of2-(tert-butoxycarbonylamino)-4-(methoxycarbonyl)phenylboronic acid (fromExample 85/Step 1) (1.0 eq.) and2-chloro-6-(4-methoxyphenethyl)nicotinonitrile (prepared from reactionof 3,5-dichloropicolinonitrile with 1-ethynyl-4-methoxybenzene followingthe procedure described in Example 44/Step 3 and reduction of theproduct following the procedure described in Example 114/Step 3) (1.0eq.), tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueoussodium carbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with EtOAc and water. The two phases wereseparated, and the aqueous layer was extracted twice with EtOAc. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vaccuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-80%EtOAc/Hexanes to give methyl5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridine-8-carboxylateas a white solid.

Step 2:(5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

To a solution of methyl5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridine-8-carboxylate(from the previous step) (1.0 eq.) in THF (0.03M) was added Super-H (10eq.) at 0° C. The solution was allowed to warm to ambient temperatureover 30 minutes. The reaction was quenched by water until no bubbling.The layers were separated and aqueous layer was extracted with EtOAc.The combined organic layer was washed with brine, dried over MgSO₄ andconcentrated en vaccuo to obtain a crude residue. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-100% EtOAc/Hexanes to give(5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanolas an off white solid (31%): ¹H NMR (acetone-d₆): δ 8.79 (d, 1H), 8.70(d, 1H), 8.35 (d, 1H), 7.61 (d, 1H), 7.33 (dd, 1H), 7.13 (d, 2H), 6.83(d, 2H), 6.62 (br s, 2H), 4.47 (s, 2H), 4.40 (br s, 1H), 3.75 (s, 3H),3.22 (t, 2H), 3.06 (t, 2H). LRMS [M+H]=360.2.

Example 96 benzo[f][1,7]naphthyridine-5,8-diamine

Step 1: tert-butyl 2-bromo-5-nitrophenylcarbamate

To a solution of 2-bromo-5-nitroaniline (commercially available) (1.0eq.) in THF (0.2M) at 0° C. under N₂ atmosphere was added dropwise 1MNaHMDS (2.5 eq.). The reaction was stirred for 15 minutes at 0° C., anda solution of di-tert-butyl dicarbonate in THF was added. The reactionwas warmed to ambient temperature overnight. The solvent was evaporated,and the resulting residue was quenched with 0.1N HCl aqueous solution.The aqueous suspension was extracted twice with EtOAc. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-30% EtOAc/Hexanesto give tert-butyl 2-bromo-5-nitrophenylcarbamate as a colorless oil.

Step 2: tert-butyl5-nitro-2-(4.4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl carbamate

Tert-butyl 2-bromo-5-nitrophenylcarbamate (from the previous step) (1.0eq.), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.8eq.), dichloro[1,1′-bis(diphenyl phosphino)ferrocene]palladium (II)(5%), and sodium acetate (4.5 eq.) were mixed in dioxane (0.2 M) underN₂ atmosphere. The reaction was heated to 100° C. and stirred overnight.The resulting suspension was cooled to ambient temperature, diluted withether, filtered through celite, and the filtrate was concentrated envaccuo. The residue was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-30% EtOAc/Hexanes to give tert-butyl5-nitro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl carbamateas a white foam.

Step 3: 8-nitrobenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-nitro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from the previous step) (1.0 eq.) and 3-bromopicolinonitrile (1.0 eq.)in toluene (0.44 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and 2N aqueouspotassium carbonate solution (2.0 eq.). The reaction was heated to 100°C. and stirred overnight. After cooling to ambient temperature, thereaction mixture was filtered to collect the precipitate. Theprecipitate was rinsed with EtOAc to give8-nitrobenzo[f][1,7]naphthyridin-5-amine as a yellow solid.

Step 4: benzo[f][1,7]naphthyridine-5,8-diamine

A mixture of 8-nitrobenzo[f][1,7]naphthyridin-5-amine (from the previousstep) (1.0 eq) and Pd/C (wet, 10% wt) in EtOH was stirred under H₂balloon overnight. The reaction mixture was diluted with DCM. Theinsoluble solid was filtered off through celite, and the filtrate wasconcentrated en vaccuo to obtain a crude residue. The crude material waswashed with acetone to give benzo[f][1,7]naphthyridine-5,8-diamine as anoff white solid: ¹H NMR (methanol-d₄): δ 8.73 (dd, 1H), 8.71 (dd, 1H),8.11 (d, 1H), 7.69 (dd, 1H), 6.86 (d, 1H), 6.82 (dd, 1H). LRMS[M+H]=211.1.

Example 97 8-(aminomethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 2-(tert-butoxycarbonylamino)-4-cyanophenylboronic acid

The titled compound was prepared according to the procedure described inExample 85/Step 1, but using 2-amino-4-cyanophenylboronic acidhydrochloride (commercially available) as the starting material. Thecrude material was purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-30% MeOH/DCM to give2-(tert-butoxycarbonylamino)-4-cyanophenylboronic acid as an off whitesolid.

Step 2: 5-aminobenzo[f][1,7]naphthyridine-8-carbonitrile

The titled compound was prepared according to the procedure described inExample 96/Step 3, but using2-(tert-butoxycarbonylamino)-4-cyanophenylboronic acid (from theprevious step) as the starting material. The crude material was rinsedwith 1:1 EtOAc/Hexanes to give5-aminobenzo[f][1,7]naphthyridine-8-carbonitrile as a pale yellow solid.

Step 4: benzo[f][1,7]naphthyridine-5,8-diamine

A mixture of 8-nitrobenzo[f][1,7]naphthyridin-5-amine (from the previousstep) (1.0 eq) and Raney Nickel (wet, 10% wt) in EtOH/ammonia (2:1) wasstirred under H₂ balloon overnight. The reaction mixture was dilutedwith DCM. The insoluble solid was filtered off through celite, and thefiltrate was concentrated en vaccuo to obtain a crude residue. The crudematerial was washed with 10% MeOH/DCM and 70% EtOAc/Hexanes to givebenzo[f][1,7]naphthyridine-5,8-diamine as an off white solid: ¹H NMR(methanol-d₄): δ 8.97 (dd, 1H), 8.90 (dd, 1H), 8.41 (d, 1H), 7.83 (dd,1H), 7.57 (d, 1H), 7.39 (dd, 1H), 3.96 (s, 2).

LRMS [M+H]=229.1.

Example 98 3-fluoro-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 3-chloro-8-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.0 eq.) and 3-bromo-6-chloropicolinonitrile(from Example 20/Step 2) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (5 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.03 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with EtOAc and water. The two phases wereseparated, and the aqueous layer was extracted twice with EtOAc. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vaccuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-40%EtOAc/Hexanes to give 3-chloro-8-methylbenzo[f][1,7]naphthyridin-5-amineas a pale yellow solid.

Step 2: 3-fluoro-8-methylbenzo[f][1,7]naphthyridin-5-amine

A mixture of 3-chloro-8-methylbenzo[f][1,7]naphthyridin-5-amine (fromthe previous step) (1.0 eq.) potassium fluoride (4.0 eq.), and18-crown-6 (0.4 eq.) in NMP (0.1M) was heated in microwave reactor at210° C. for 2 hours. After cooling to ambient temperature, the reactionresidue was purified by flash chromatography on a COMBIFLASH® system(ISCO) using 0-30% EtOAc/Hexanes to give3-fluoro-8-methylbenzo[f][1,7]naphthyridin-5-amine as a white solid. ¹HNMR (acetone-d₆): δ 9.20 (dd, 1H), 8.32 (d, 1H), 7.58 (dd, 1H), 7.46 (d,1H), 7.21 (dd, 1H), 6.51 (br s, 2H), 2.47 (s, 3H). LRMS [M+H]=228.1.

Example 99 (5-amino-3-fluorobenzo[f][1,7]naphthyridin-8-yl)methanol

Step 1: tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

The titled compound was prepared according to the procedure described inExample 93/Step 1 and 2, but using5-((tert-butyldimethylsilyloxy)methyl)-2-chloroaniline (commerciallyavailable) as the starting material. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-20%EtOAc/Hexanes to give tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamateas a white foam.

Step 2:8-((tert-butyldimethylsilyloxy)methyl)-3-chlorobenzo[f][1,7]naphthyridin-5-amine

The titled compound was prepared according to the procedure described inExample 98/Step 1, but using tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from the previous step) as the starting material. The crude materialwas purified by flash chromatography on a COMBIFLASH® system (ISCO)using 0-15% EtOAc/Hexanes to give8-((tert-butyldimethylsilyloxy)methyl)-3-chlorobenzo[f][1,7]naphthyridin-5-amineas a pale yellow solid.

Step 3: (5-amino-3-chlorobenzo[f][1,7]naphthyridin-8-yl)methanol

A solution of8-((tert-butyldimethylsilyloxy)methyl)-3-chlorobenzo[f][1,7]naphthyridin-5-amine(from the previous step) (1.0 eq.) and TBAF (1.1 eq.) in THF was stirredat ambient temperature overnight. The reaction was quenched withsaturated NaHCO₃. The two phases were separated, and the aqueous layerwas extracted twice with Et₂O. The combined organic layers were washedwith brine, dried over anhydrous MgSO₄, and concentrated en vaccuo. Thecrude material was purified by flash chromatography on a COMBIFLASH®system (ISCO) using 0-5% MeOH/DCM to give(5-amino-3-chlorobenzo[f][1,7]naphthyridin-8-yl)methanol as a whitesolid.

Step 4: (5-amino-3-fluorobenzo[f][1,7]naphthyridin-8-yl)methanol

The titled compound was prepared according to the procedure described inExample 98/Step 2, but using(5-amino-3-chlorobenzo[f][1,7]naphthyridin-8-yl)methanol (from theprevious step) as the starting material. The crude material was purifiedby flash chromatography on a COMBIFLASH® system (ISCO) using 0-40%EtOAc/Hexanes to give(5-amino-3-fluorobenzo[f][1,7]naphthyridin-8-yl)methanol as a whitesolid. ¹H NMR (methanol-d₄): δ 9.15 (dd, 1H), 8.38 (d, 1H), 7.64 (d,1H), 7.55 (dd, 1H), 7.41 (dd, 1H), 4.77 (s, 2H).

LRMS [M+H]=244.1.

Example 100 3-chlorobenzo[f][1,7]naphthyridine-5,8-diamine

Step 1: 3-chloro-8-nitrobenzo[f][1,7]naphthyridin-5-amine

The titled compound was prepared according to the procedure described inExample 98/Step 1, but using tert-butyl5-nitro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(commercially available) as the starting material. The crude materialwas purified by flash chromatography on a COMBIFLASH® system (ISCO)using 0-40% EtOAc/Hexanes to give3-chloro-8-nitrobenzo[f][1,7]naphthyridin-5-amine as a pale yellowsolid.

Step 2: 3-chlorobenzo[f][1,7]naphthyridine-5,8-diamine

A mixture of 8-nitrobenzo[f][1,7]naphthyridin-5-amine (from the previousstep) (1.0 eq) and Raney Nickel (wet, 10% wt) in EtOH was stirred underH₂ balloon overnight. The reaction mixture was diluted with DCM. Theinsoluble solid was filtered off through celite, and the filtrate wasconcentrated en vaccuo to obtain a crude residue. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-100% EtOAc/Hexanes to give3-chlorobenzo[f][1,7]naphthyridine-5,8-diamine as a white solid. ¹H NMR(methanol-d₄): δ 8.75 (d, 1H), 8.08 (dd, 1H), 7.70 (d, 1H), 6.84-6.81(m, 2H). LRMS [M+H]=245.1.

Example 101 3-fluorobenzo[f][1,7]naphthyridine-5,8-diamine

The titled compound was prepared according to the procedure described inExample 98/Step 2, but using3-chlorobenzo[f][1,7]naphthyridine-5,8-diamine (from Example 100) as thestarting material. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-7% MeOH/DCM togive 3-fluorobenzo[f][1,7]naphthyridine-5,8-diamine as a white solid. ¹HNMR (methanol-d₄): δ 8.93 (dd, 1H), 8.09 (d, 1H), 7.44 (dd, 1H),6.86-6.83 (m, 2H). LRMS [M+H]=229.1.

Example 102 8-isobutylbenzo[f][1,7]naphthyridin-5-amine

8-Isobutylbenzo[f][1,7]naphthyridin-5-amine was prepared from5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde (from Example 87) withisopropyl(triphenyl)phosphonium bromide following the proceduresdescribed for Example 91 (wittig reaction) and Example 92 (reduction).¹H NMR (acetone-d₆): δ 8.98 (dd, 1H), 8.88 (dd, 1H), 8.35 (d, 1H), 7.82(dd, 1H), 7.44 (d, 1H), 7.18 (dd, 1H), 6.73 (br s, 2H), 2.63 (d, 2H),2.04-1.94 (m, 1H), 0.94 (d, 6H). LRMS [M+H]=252.1.

Example 103 (E)-8-(prop-1-enyl)benzo[f][1,7]naphthyridin-5-amine

(E)-8-(prop-1-enyl)benzo[f][1,7]naphthyridin-5-amine was prepared from5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde (from Example 87) withethyl(triphenyl)phosphonium bromide following the procedures describedfor Example 91. ¹H NMR (acetone-d₆): δ 8.98 (dd, 1H), 8.88 (dd, 1H),8.36 (d, 1H), 7.83 (dd, 1H), 7.54 (d, 1H), 7.43 (dd, 1H), 6.67 (br s,2H), 6.60-6.42 (m, 2H), 1.92 (dd, 3H). LRMS [M+H]=236.1.

Example 104 8-propylbenzo[f][1,7]naphthyridin-5-amine

8-Propylbenzo[f][1,7]naphthyridin-5-amine was prepared from(E)-8-(prop-1-enyl)benzo[f][1,7]naphthyridin-5-amine (from Example 103)following the procedures described for Example 92. ¹H NMR (acetone-d₆):δ 8.99 (dd, 1H), 8.88 (dd, 1H), 8.35 (d, 1H), 7.83 (dd, 1H), 7.45 (d,1H), 7.21 (dd, 1H), 6.64 (br s, 2H), 2.74 (t, 2H), 1.74 (qt, 2H), 0.98(t, 3H). LRMS [M+H]=238.1.

Example 105 8-(2-cyclopropylethyl)benzo[f][1,7]naphthyridin-5-amine

8-(2-Cyclopropylethyl)benzo[f][1,7]naphthyridin-5-amine was preparedfrom 5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde (from Example 87)with (cyclopropylmethyl)triphenylphosphonium bromide following theprocedures described for Example 91 (wittig reaction) and Example 92(reduction). ¹H NMR (acetone-d₆): δ 8.99 (dd, 1H), 8.88 (dd, 1H), 8.35(d, 1H), 7.83 (dd, 1H), 7.47 (d, 1H), 7.23 (dd, 1H), 6.64 (br s, 2H),1.60 (q, 2H), 1.34-1.25 (m, 1H), 0.91-0.72 (m, 2H), 0.45-0.41 (m, 2H),0.11-0.07 (m, 2H). LRMS [M+H]=264.1.

Example 106 8-phenethylbenzo[f][1,7]naphthyridin-5-amine

8-Phenethylbenzo[f][1,7]naphthyridin-5-amine was prepared from5-aminobenzo[f][1,7]naphthyridine-8-carbaldehyde (from Example 87) withbenzyltriphenylphosphonium bromide following the procedures describedfor Example 91 (wittig reaction) and Example 92 (reduction). ¹H NMR(acetone-d₆): δ 8.99 (dd, 1H), 8.88 (dd, 1H), 8.35 (d, 1H), 7.83 (dd,1H), 7.49 (d, 1H), 7.29-7.15 (dd, 6H), 6.70 (br s, 2H), 3.10-3.00 (m,4H). LRMS [M+H]=300.1.

Example 107(5-amino-2-(4-bromophenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

(5-Amino-2-(4-bromophenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol wasprepared from(5-amino-2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol(from Example 95) following the procedures described for Example 86. ¹HNMR (acetone-d₆): δ 8.81 (d, 1H), 8.72 (d, 1H), 8.40 (d, 1H), 7.68 (d,1H), 7.39 (dd, 1H), 7.08 (d, 2H), 6.74 (d, 2H), 6.66 (br s, 2H), 4.49(s, 2H), 3.21 (t, 2H), 3.03 (t, 2H). LRMS [M+H]=408.1.

Example 108(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanolwas prepared from tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(Example 99/Step 1) and3-chloro-5-((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile (fromExample 49/Step 1) following the procedures described for Example44/Step 4 and deprotection of TBS group following the proceduredescribes from Example 99/Step 3. ¹H NMR (acetone-d₆): δ 8.79 (s, 1H),8.73 (s, 1H), 8.35 (d, 1H), 7.61 (s, 1H), 7.33 (d, 1H), 7.09 (d, 1H),6.75 (d, 1H), 6.68 (dd, 1H), 6.57 (br s, 2H), 4.47 (d, 2H), 4.32 (t,1H), 3.58 (s, 3H), 3.17 (t, 2H), 3.04 (t, 2H), 2.30 (s, 3H). LRMS[M+H]=374.2.

Example 1092-(4-methoxy-2-methylphenethyl)-8-pentylbenzo[f][1,7]naphthyridin-5-amine

and Example 1108-(2-cyclopropylethyl)-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: tert-butyl 5-bromo-2-chlorophenylcarbamate

The titled compound was prepared according to the procedure described inExample 5/Step 1, but using 5-bromo-2-chloroaniline (commerciallyavailable) as the starting material. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-40%EtOAc/Hexanes to give tert-butyl 5-bromo-2-chlorophenylcarbamate as apale yellow solid.

Step 2: (E)-tert-butyl 2-chloro-5-(2-cyclopropylvinyl)phenylcarbamate

A solution of tert-butyl 5-bromo-2-chlorophenylcarbamate (from theprevious step) (1.0 eq.) and(E)-2-(2-cyclopropylvinyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(commercially available) (1.0 eq.) in toluene (0.2 M) was mixed withtetrakis(triphenyl-phosphine)palladium (5 mol %) and 2N aqueouspotassium carbonate solution (2.0 eq.). The reaction was heated to 100°C. and stirred overnight. After cooling to ambient temperature, thereaction mixture was diluted with EtOAc and water. The two phases wereseparated, and the aqueous layer was extracted twice with EtOAc. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vaccuo. The crude product was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-5%EtOAc/Hexanes to give (E)-tert-butyl2-chloro-5-(2-cyclopropylvinyl)phenylcarbamate as a pale yellow solid.

Step 3: (E)-tert-butyl5-(2-cyclopropylvinyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate

The titled compound was prepared according to the procedure described inExample 93/Step 2, but using (E)-tert-butyl2-chloro-5-(2-cyclopropylvinyl)phenylcarbamate (from previous step) asthe starting material. The crude product was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-10% EtOAc/Hexanesto give (E)-tert-butyl5-(2-cyclopropylvinyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamateas a pale yellow solid.

Step 4:2-(4-methoxy-2-methylphenethyl)-8-pentylbenzo[f][1,7]naphthyridin-5-amineand8-(2-cyclopropylethyl)-2-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine

The titled compounds were prepared according to the procedure describedin Example 44/Step 4 (Suzuki coupling) and 5 (reduction), but using(E)-tert-butyl5-(2-cyclopropylvinyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from previous step) and3-chloro-5-((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile (fromExample 49/Step 1) as the starting material. The crude product waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-40% EtOAc/Hexanes to give Example 109 as a white solid: ¹H NMR(acetone-d₆): δ 8.76 (d, 1H), 8.70 (d, 1H), 8.29 (d, 1H), 7.44 (d, 1H),7.18 (dd, 1H), 7.08 (d, 1H), 6.74 (d, 1H), 6.68 (dd, 1H), 6.59 (br s,2H), 3.74 (s, 3H), 3.18 (t, 2H), 3.04 (t, 2H), 2.75 (t, 2H), 2.29 (s,3H), 1.75-1.68 (m, 2H), 1.40-1.35 (m, 4H), 0.90 (s, 3H); LRMS[M+H]=414.3; and Example 110 as an off white solid: ¹H NMR (acetone-d₆):δ 8.76 (d, 1H), 8.70 (d, 1H), 8.28 (d, 1H), 7.45 (d, 1H), 7.19 (dd, 1H),7.08 (d, 1H), 6.74 (d, 1H), 6.67 (dd, 1H), 6.55 (br s, 2H), 3.73 (s,3H), 3.16 (t, 2H), 3.03 (t, 2H), 2.29 (s, 3H), 1.60 (q, 2H), 1.29-1.28(m, 1H), 0.89-0.74 (m, 2H), 0.44-0.41 (m, 2H), 0.10-0.07 (m, 2H). LRMS[M+H]=412.3.

Example 111(5-amino-2-(2,4,6-trimethylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

(5-Amino-2-(2,4,6-trimethylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanolwas prepared from tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 99/step 1), 2-ethynyl-1,3,5-trimethylbenzene (commerciallyavailable) and 3-chloro-5-(mesitylethynyl)picolinonitrile (from Example77/step 1) following the procedures described for Example 44/Step 4,Example 99/step 3 (deprotection of TBS) and Example 77/step 3(reduction). ¹H NMR (acetone-d₆): δ 8.77 (s, 2H), 8.34 (d, 1H), 7.61 (s,1H), 7.33 (d, 1H), 6.84 (s, 2H), 6.60 (br s, 2H), 4.77 (d, 2H), 4.35 (t,1H), 3.08 (s, 3H), 2.84 (s, 6H), 2.30-2.29 (m, 4H). LRMS [M+H]=372.2.

Example 112(5-amino-2-(4-propoxyphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

(5-Amino-2-(4-propoxyphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanolwas prepared from8-((tert-butyldimethylsilyloxy)methyl)-3-chlorobenzo[f][1,7]naphthyridin-5-amine(from Example 99/step 1) and3-chloro-5-(4-propoxyphenethyl)picolinonitrile (from Example 79/step 2)following the procedures described for Example 44/Step 4 and Example99/step 3 (deprotection of TBS). ¹H NMR (acetone-d₆): δ 8.79 (d, 1H),8.70 (d, 1H), 8.35 (d, 1H), 7.61 (d, 1H), 7.33 (dd, 1H), 7.17 (d, 2H),6.83 (d, 2H), 6.57 (br s, 2H), 4.77 (d, 2H), 4.34 (t, 1H), 3.89 (t, 2H),3.22 (t, 2H), 3.06 (t, 2H), 1.83-1.70 (m, 2H), 1.00 (t, 3H). LRMS[M+H]=388.2.

Example 113(2-(2-(1H-indol-5-yl)ethyl)-5-aminobenzo[f][1,7]naphthyridin-8-yl)methanol

(2-(2-(1H-indol-5-yl)ethyl)-5-aminobenzo[f][1,7]naphthyridin-8-yl)methanolwas prepared from tert-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 99/step 1) and5-((1H-indol-5-yl)ethynyl)-3-chloropicolinonitrile (from Example 44/step3) following the procedures described for Example 44/Step 4 and Example99/step 3 (deprotection of TBS). ¹H NMR (acetone-d₆): δ 10.19 (t, 1H),8.83 (d, 1H), 8.71 (d, 1H), 8.35 (d, 1H), 7.60 (d, 1H), 7.46 (d, 1H),7.36-7.27 (m, 3H), 7.04 (dd, 1H), 6.57 (br s, 2H), 6.38 (dt, 1H), 4.77(d, 2H), 4.36 (t, 1H), 3.29 (t, 2H), 3.19 (t, 2H). LRMS [M+H]=369.2.

Example 114N-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)acetamide

Step 1: N-(4-ethynylphenyl)acetamide

To a solution of 4-ethynylaniline (commercially available) (1.0 eq.),and triethylamine (1.0 eq.) in methylene chloride (0.04 M), acetylchloride (1.5 eq.) was added slowly. Then the reaction mixture wasstirred at 0° C. for 1 hour. After warmed to ambient temperature, thereaction mixture was diluted with ethyl acetate and water. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to give N-(4-ethynylphenyl)acetamide as awhite solid.

Step 2: N-(4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)phenyl)acetamide

A solution of 3,5-dichloropicolinonitrile (1.0 eq.),N-(4-ethynylphenyl)acetamide (from the previous step) (1.0 eq.),bis(triphenyl-phosphine)palladium chloride (10 mol %), copper iodide (10mol %), and triethylamine (5.0 eq.) in DMF (0.04 M) was stirred at 60°C. for 4 hours. After cooling to ambient temperature, the reactionmixture was diluted with ethyl acetate and water. The two phases wereseparated. The organic layer was washed twice with water, dried overanhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to giveN-(4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)phenyl)acetamide as a whitesolid.

Step 3: N-(4-(2-(5-chloro-6-cyanopyridin-3-yl)ethyl)phenyl)acetamide

To a solution ofN-(4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)phenyl)acetamide (from theprevious step) in ethyl acetate/methanol (1:4, 0.05 M) was added 10% wtpalladium on carbon (0.2 eq.). Hydrogen gas was introduced via aballoon, and the reaction was stirred for 3 hours. The mixture wasfiltered through a pad of celite, washing with dichloromethane. Thefiltrate was concentrated en vaccuo and purified by a COMBIFLASH® system(ISCO) using 0-80% ethyl acetate in hexane to giveN-(4-(2-(5-chloro-6-cyanopyridin-3-yl)ethyl)phenyl)acetamide.

Step 4:N-(4-O-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)acetamide

A solution ofN-(4-(2-(5-chloro-6-cyanopyridin-3-yl)ethyl)phenyl)acetamide (from theprevious step) (1.0 eq.), tert-butyl4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 5/Step 2) (1.5 eq.),Tris(dibenzylideneacetone)dipalladium(0) (10 mol %),dicyclohexyl(2′,6′-dimethoxybiphenyl-2-yl)phosphine (20 mol %), andpotassium phosphate (2.0 eq.) in n-butanol/H₂O (2.5:1, 0.04 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and water. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to giveN-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)acetamideas a white solid. ¹H NMR (CDCl₃): δ 8.51 (s, 1H), 8.32 (s, 1H), 8.01 (d,1H), 7.44 (s, 1H), 7.33-7.36 (m, 2H), 7.03-7.19 (m, 3H), 5.98 (br, 2H),3.07-3.11 (m, 2H), 2.94-2.98 (m, 2H), 2.44 (s, 3H), 2.10 (s, 3H). LRMS[M+H]=371.2.

Example 115 methyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate

Step 1: methyl 3-methyl-4-((triethylsilyl)ethynyl)benzoate

A solution of methyl 4-bromo-3-methylbenzoate (1.0 eq.),triethyl(ethynyl)silane (1.0 eq.), bis(triphenyl-phosphine)palladiumchloride (10 mol %), copper iodide (10 mol %), and triethylamine (5.0eq.) in DMF (0.04 M) was stirred at 60° C. for 4 hours. After cooling toambient temperature, the reaction mixture was diluted with ethyl acetateand water. The two phases were separated. The organic layer was washedtwice with water, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-50% ethyl acetate in hexane to givemethyl 3-methyl-4-((triethylsilyl)ethynyl)benzoate as a white solid.

Step 2: methyl 4-ethynyl-3-methylbenzoate

To a solution of methyl 3-methyl-4-((triethylsilyl)ethynyl)benzoate(from the previous step) (1.0 eq.) in THF (0.2 M), was added TBAF (0.2eq.) slowly at 0° C. Then the reaction mixture was stirred at 0° C. for1 hour. After warmed to ambient temperature, the reaction mixture wasdiluted with ethyl acetate and water. The two phases were separated, andthe aqueous layer was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-80% ethyl acetatein hexane to give methyl 4-ethynyl-3-methylbenzoate as a white solid.

Step 3: methyl4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)-3-methylbenzoate

A solution of 3,5-dichloropicolinonitrile (1.0 eq.), methyl4-ethynyl-3-methylbenzoate (from the previous step) (1.0 eq.),bis(triphenyl-phosphine)palladium chloride (10 mol %), copper iodide (10mol %), and triethylamine (5.0 eq.) in DMF (0.04 M) was stirred at 60°C. for 4 hours. After cooling to ambient temperature, the reactionmixture was diluted with ethyl acetate and water. The two phases wereseparated. The organic layer was washed twice with water, dried overanhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to give methyl4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)-3-methylbenzoate as a whitesolid.

Step 4: methyl methyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)-3-methylbenzoate

A solution of methyl4-((5-chloro-6-cyanopyridin-3-yl)ethynyl)-3-methylbenzoate (from theprevious step) (1.0 eq.), tert-butyl4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(From Example 5/Step 2) (1.5 eq.),tris(dibenzylideneacetone)dipalladium(0) (10 mol %),dicyclohexyl(2′,6′-dimethoxybiphenyl-2-yl)phosphine (20 mol %), andpotassium phosphate (2.0 eq.) in n-butanol/H₂O (2.5:1, 0.04 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with ethyl acetate and water. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane to give methylmethyl-4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)-3-methylbenzoateas a white solid.

Step 5: methyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate

To a solution of methyl methyl4-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)-3-methylbenzoate(from the previous step) in ethyl acetate/methanol (1:4, 0.05 M) wasadded 10% wt palladium on carbon (0.2 eq.). Hydrogen gas was introducedvia a balloon, and the reaction was stirred for 3 hours. The mixture wasfiltered through a pad of celite, washing with dichloromethane. Thefiltrate was concentrated en vaccuo and purified by a COMBIFLASH® system(ISCO) using 0-80% ethyl acetate in hexane to give methyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate.¹H NMR (CDCl₃): δ 8.61 (s, 1H), 8.40 (s, 1H), 8.09 (d, 1H), 7.83 (s,1H), 7.81 (d, 1H), 7.54 (s, 1H), 7.18-7.20 (m, 2H), 6.17 (br, 2H), 3.92(s, 3H), 3.10-3.16 (m, 4H), 2.53 (s, 3H), 2.36 (s, 3H). LRMS[M+H]=386.2.

Example 1164-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N,3-dimethylbenzamide

Step 1:4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoicacid

A solution of methyl4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate(from Example 115) (1.0 eq.), and 1N sodium hydroxide (1.5 eq.) inmethanol (0.04 M) was stirred at 60° C. for 4 hours. After cooling toambient temperature, the reaction mixture was diluted with ethyl acetateand water. The two phases were separated. The organic layer was washedtwice with water, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-50% ethyl acetate in hexane to give4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoicacid as a white solid.

Step 2:4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride

A solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoicacid (from the previous step) in thionyl chloride was stirred at 60° C.for 3 hour. After cooling to ambient temperature, the reaction mixturewas concentrated en vaccuo. The crude material was used for next stepwithout purification.

Step 3:4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N,3-dimethylbenzamide

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (from the previous step) (Example 5) and triethylamine (2.5eq.) in ether (0.05 M) was added methanamine (5.0 eq.). The reactionmixture was stirred for overnight. Then the reaction mixture was dilutedwith ethyl acetate and water. The two phases were separated, and theaqueous layer was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-80% ethyl acetatein hexane to give4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N,3-dimethylbenzamideas a white solid. ¹H NMR (CDCl₃): δ 8.62 (s, 1H), 8.32 (s, 1H), 8.04 (d,1H), 7.60 (s, 1H), 7.46-7.52 (m, 2H), 7.09-7.11 (m, 2H), 6.05 (br, 2H),3.09-3.17 (m, 4H), 3.00 (d, 3H), 2.52 (s, 3H), 2.33 (s, 3H). LRMS[M+H]=385.2.

Example 1174-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-N,3-dimethylbenzamide

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and triethylamine (2.5 eq.) in ether (0.05M) was added N¹,N¹,N²-trimethylethane-1,2-diamine (5.0 eq.). Thereaction mixture was stirred for overnight. Then the reaction mixturewas diluted with ethyl acetate and water. The two phases were separated,and the aqueous layer was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄, and concentrated en vaccuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-80% ethylacetate in hexane to give4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-N,3-dimethylbenzamideas a white solid. ¹H NMR (CDCl₃): δ 8.66 (s, 1H), 8.37 (s, 1H), 8.07 (d,1H), 7.63 (s, 1H), 7.09-7.30 (m, 4H), 3.90 (br, 2H), 3.01-3.19 (m, 4H),3.08 (s, 6H), 2.72 (br, 5H), 2.52 (s, 3H), 2.33 (s, 3H). LRMS[M+H]=456.3.

Example 118 2-(4-methoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine

2-(4-Methoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine was preparedfrom 1-ethynyl-4-methoxybenzene (Example 116/Step 2) following theprocedures described for Example 45/Steps 1 to 3. ¹H NMR (CDCl₃): δ 8.69(s, 1H), 8.47 (s, 1H), 8.27 (d, 1H), 7.80 (d, 2H), 7.58-7.66 (m, 1H),7.33-7.42 (m, 1H), 7.15 (d, 2H), 6.90 (d, 2H), 6.25 (br, 2H), 3.86 (s,3H), 3.13-3.23 (m, 2H), 2.97-3.10 (m, 2H). LRMS [M+H]=330.2.

Example 1192-(4-methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine

2-(4-Methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine wasprepared from 1-ethynyl-4-methoxy-2-methylbenzene (commerciallyavailable) following the procedures described for Example 45/Step 1 to3. ¹H NMR (CDCl₃): δ 8.60 (s, 1H), 8.37 (s, 1H), 8.18 (d, 1H), 7.69 (d,1H), 7.49-7.57 (m, 1H), 7.24-7.34 (m, 1H), 6.98 (d, 1H), 6.56-6.70 (m,2H), 6.00 (br, 2H), 3.70 (s, 3H), 3.00-3.09 (m, 2H), 2.83-2.96 (m, 2H),2.20 (s, 3H). LRMS [M+H]=344.2.

Example 1204-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and ammonia following the proceduresdescribed for Example 117. ¹H NMR (CDCl₃): δ 8.60 (s, 1H), 8.35 (s, 1H),8.05 (d, 1H), 7.65 (s, 1H), 7.51-7.53 (m, 2H), 7.13-7.21 (m, 2H),3.09-3.16 (m, 4H), 2.51 (s, 3H), 2.34 (s, 3H). LRMS [M+H]=371.2

Example 1214-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N,N,3-trimethylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N,N,3-trimethylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and dimethylamine following the proceduresdescribed for Example 117. ¹H NMR (CDCl₃): δ 8.68 (s, 1H), 8.32 (s, 1H),8.04 (d, 1H), 7.66 (s, 1H), 7.31 (d, 1H), 7.06-7.18 (m, 3H), 3.08-3.19(m, 4H), 2.96 (d, 3H), 2.54 (s, 3H), 2.33 (s, 3H), 2.05 (s, 3H). LRMS[M+H]=399.2

Example 1224-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-hydroxyethyl)-3-methylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-hydroxyethyl)-3-methylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and 2-aminoethanol following theprocedures described for Example 117. ¹H NMR (CDCl₃): δ 8.59 (s, 1H),8.34 (s, 1H), 8.04 (d, 1H), 7.50-7.62 (m, 3H), 7.08-7.25 (m, 2H), 3.80(t, 2H), 3.63 (t, 2H), 3.07-3.16 (m, 4H), 2.51 (s, 3H), 2.32 (s, 3H).LRMS [M+H]=415.2

Example 1234-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-3-methylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-3-methylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and N¹,N¹-dimethylethane-1,2-diaminefollowing the procedures described for Example 117. ¹H NMR(methanol-d₄): δ 8.60 (s, 1H), 8.39 (s, 1H), 8.08 (d, 1H), 7.68 (s, 1H),7.57-7.59 (m, 2H), 7.19-7.22 (m, 2H), 3.57-3.61 (m, 2H), 3.07-3.16 (m,4H), 2.64-2.67 (m, 2H), 2.52 (s, 3H), 2.38 (s, 6H), 2.35 (s, 3H). LRMS[M+H]=442.3

Example 124(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(pyrrolidin-1-yl)methanone

(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(pyrrolidin-1-yl)methanonewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and pyrrolidine following the proceduresdescribed for Example 117. ¹H NMR (methanol-d₄): δ 8.60 (s, 1H), 8.42(s, 1H), 8.09 (d, 1H), 7.34 (s, 1H), 7.23 (s, 1H), 7.05-7.15 (m, 3H),3.49 (t, 2H), 3.27 (t, 2H), 3.05-3.17 (m, 4H), 2.42 (s, 3H), 2.26 (s,3H), 1.88-1.91 (m, 2H), 1.73-1.77 (m, 2H). LRMS [M+H]=425.2

Example 1254-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(diethylamino)ethyl)-3-methylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(diethylamino)ethyl)-3-methylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and N¹,N¹-diethylethane-1,2-diaminefollowing the procedures described for Example 117. ¹H NMR(methanol-d₄): δ 8.55 (s, 1H), 8.48 (s, 1H), 8.10 (d, 1H), 7.56 (s, 1H),7.47-7.50 (m, 1H), 7.33 (s, 1H), 7.10-7.14 (m, 2H), 3.44 (t, 2H), 3.25(t, 2H), 3.08-3.14 (m, 4H), 2.62-2.72 (m, 4H), 2.42 (s, 3H), 2.27 (s,3H), 1.05 (t, 6H). LRMS [M+H]=470.3

Example 126(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(4-ethylpiperazin-1-yl)methanone

(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(4-ethylpiperazin-1-yl)methanonewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and 1-ethylpiperazine following theprocedures described for Example 117. ¹H NMR (Methanol-d₄): δ 8.59 (s,1H), 8.37 (s, 1H), 8.06 (d, 1H), 7.32 (s, 1H), 7.00-7.12 (m, 4H), 3.67(br, 2H), 3.06-3.13 (m, 4H), 2.45 (br, 4H), 2.37 (q, 2H), 2.41 (s, 3H),2.26 (s, 3H), 2.19 (br, 2H), 1.04 (t, 3H). LRMS [M+H]=468.3

Example 127(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(piperazin-1-yl)methanone

(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)(piperazin-1-yl)methanonewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and piperazine following the proceduresdescribed for Example 117. ¹H NMR (methanol-d₄): δ 8.66 (s, 1H), 8.55(s, 1H), 8.19 (d, 1H), 7.38 (s, 1H), 7.21-7.23 (m, 2H), 7.10-7.15 (m,2H), 3.66 (br, 6H), 3.08-3.18 (m, 6H), 2.45 (s, 3H), 2.30 (s, 3H). LRMS[M+H]=440.2

Example 1284-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methyl-N-(2-(pyrrolidin-1-yl)ethyl)benzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methyl-N-(2-(pyrrolidin-1-yl)ethyl)benzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and 2-(pyrrolidin-1-yl)ethanaminefollowing the procedures described for Example 117. ¹H NMR (CDCl₃): δ8.58 (s, 1H), 8.38 (s, 1H), 8.07 (d, 1H), 7.64 (s, 1H), 7.51-7.55 (m,2H), 7.12-7.20 (m, 2H), 6.26 (br, 2H), 3.61 (dd, 2H), 3.05-3.12 (m, 4H),2.81 (t, 2H), 2.69 (br, 4H), 2.50 (s, 3H), 2.33 (s, 3H), 1.83-1.85 (m,4H). LRMS [M+H]=468.3

Example 1294-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-aminoethyl)-3-methylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-aminoethyl)-3-methylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and ethane-1,2-diamine following theprocedures described for Example 117. ¹H NMR (CDCl₃): δ 8.59 (s, 1H),8.37 (s, 1H), 8.07 (d, 1H), 7.63 (s, 1H), 7.51 (br, 2H), 7.12-7.21 (m,2H), 6.25 (br, 2H), 3.48-3.52 (m, 2H), 3.08-3.15 (m, 4H), 2.94 (t, 2H),2.51 (s, 3H), 2.34 (s, 3H). LRMS [M+H]=414.2

Example 1304-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-N,3-dimethylbenzamide

4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-N,3-dimethylbenzamidewas prepared from4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and N¹,N¹,N²-trimethylethane-1,2-diaminefollowing the procedures described for Example 117. ¹H NMR(methanol-d₄): δ 8.84 (s, 1H), 8.63 (s, 1H), 8.39 (d, 1H), 7.76-7.83 (m,2H), 7.60-7.64 (m, 1H), 7.37 (s, 1H), 7.19-7.29 (m, 2H), 3.96 (t, 2H),3.48 (t, 2H), 3.32 (t, 2H), 3.20 (t, 2H), 3.09 (s, 3H), 3.06 (s, 6H),2.42 (s, 3H). LRMS [M+H]=442.3

Example 1314-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-N-methylbenzamide

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-(dimethylamino)ethyl)-N-methylbenzamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and N¹,N¹,N²-trimethylethane-1,2-diaminefollowing the procedures described for Example 117. ¹H NMR (CDCl₃): δ8.64 (s, 1H), 8.36 (s, 1H), 8.05 (d, 1H), 7.60 (s, 1H), 7.41 (d, 2H),7.31 (d, 1H), 7.21 (d, 2H), 3.91 (t, 2H), 3.44 (t, 2H), 3.25 (t, 2H),3.12 (t, 2H), 3.03 (s, 3H), 3.01 (s, 6H), 2.53 (s, 3H). LRMS [M+H]=442.3

Example 1322-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol

2-(4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-olwas prepared following the procedures described for Example 78, butusing methyl4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoatewhich was prepared analogous to Example 115 but using tert-butyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate in Step4. LRMS [M+H]=372.2

Example 1332-(4-butoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-Butoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine wasprepared following the procedures described for Example 45/Steps 1 to 3,but using 1-butoxy-4-ethynylbenzene (commercially available) with3,5-dichloropicolinonitrile (commercially available) in step 1. ¹H NMR(Acetone-d₆): δ 8.75 (s, 1H), 8.68 (s, 1H), 8.28 (d, 1H), 7.42 (s, 1H),7.10-7.18 (m, 3H), 6.84 (d, 2H), 6.58 (br, 2H), 3.94 (t, 2H), 3.21 (t,2H), 3.05 (t, 2H), 2.46 (s, 3H), 1.65-1.75 (m, 2H), 1.41-1.58 (m, 2H),0.94 (s, 3H). LRMS [M+H]=386.2.

Example 1342-(2-(biphenyl-4-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(2-(Biphenyl-4-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine wasprepared following the procedures described for Example 45/Steps 1 to 3,but using 4-ethynylbiphenyl (commercially available) with3,5-dichloropicolinonitrile (commercially available) in Step 1. ¹H NMR(Acetone-d₆): δ 8.80 (s, 1H), 8.75 (s, 1H), 8.26 (d, 2H), 7.55-7.69 (m,4H), 7.30-7.46 (m, 4H), 7.13 (d, 2H), 6.58 (br, 2H), 3.30 (t, 2H), 3.18(t, 2H), 2.45 (s, 3H).

LRMS [M+H]=390.2

Example 1352-((1,3-dihydroisobenzofuran-1-yl)methyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1:2-((5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)methanol

2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)methanolwas prepared following the procedures described for Example 45/Steps 1to 2, but using (2-ethynylphenyl)methanol (commercially available) with3,5-dichloropicolinonitrile (commercially available) in Step 1.

Step 2:2-(1,3-dihydroisobenzofuran-1-yl)methyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a solution of2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethynyl)phenyl)methanol(1.0 equiv.) (from the previous step) in ethanol (0.05 M) was added 10%wt palladium on carbon (0.2 equiv. by weight). Hydrogen gas was thenintroduced via a balloon, and the reaction was allowed to stir for 18hours. At this point, the mixture was filtered through a pad of celite,washing with methanol. The volatiles were removed in vacuo and theresulting residue was purified by a COMBIFLASH® system (ISCO) using0-60% ethyl acetate in hexanes to give2-(1,3-dihydroisobenzofuran-1-yl)methyl)-8-methylbenzo[f][1,7]naphthyridin-5-amineas a solid. ¹H NMR (Acetone-d₆): δ 8.78 (s, 1H), 8.74 (s, 1H), 8.24 (d,2H), 7.40-7.44 (m, 2H), 7.20-7.34 (m, 3H), 6.61 (br, 2H), 5.63-5.69 (m,1H), 4.89-5.00 (dd, 2H), 3.51-3.56 (dd, 1H), 3.28-3.34 (dd, 1H), 2.46(s, 3H). LRMS [M+H]=342.1

Example 1368-methyl-2-(4-(2-methylalkyloxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) (1.0 equiv.) in dimethylformamide (0.10 M) was addedanhydrous potassium carbonate (1.5 euiv.) followed by methalkyl bromide(1.2 equiv.). The resulting mixture was allowed to stir for 18 hours at100° C. After cooling to ambient temperature, the mixture was dilutedwith ethyl acetate and water. The biphasic layers were separated and theaqueous layer was washed twice with ethyl acetate. The combined organiclayers were dried over anhydrous Na₂SO₄ and the volatiles were removedin vacuo. The resulting residue was purified by a COMBIFLASH® system(ISCO) using 0-60% ethyl acetate in hexanes to provide8-methyl-2-(4-(2-methylalkyloxy)phenethyl)benzo[f][1,7]naphthyridin-5-amineas a solid. ¹H NMR (Acetone-d₆): δ 8.75 (s, 1H), 8.68 (s, 1H), 8.27 (d,1H), 7.41 (s, 1H), 7.12-7.19 (m, 3H), 6.87 (d, 2H), 6.60 (br, 2H), 5.06(s, 1H), 4.93 (s, 1H), 4.43 (s, 2H), 3.20 (t, 2H), 3.05 (t, 2H), 2.45(s, 3H), 1.79 (s, 3H). LRMS [M+H]=384.2

Example 1372-(4-(isopentyloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(Isopentyloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 136, butusing 1-bromo-3-methylbutane. ¹H NMR (Acetone-d₆): δ 8.72 (s, 1H), 8.69(s, 1H), 8.26 (d, 1H), 7.43 (s, 1H), 7.12-7.18 (m, 3H), 6.84 (d, 2H),6.50 (br, 2H), 3.98 (t, 2H), 3.21 (t, 2H), 3.06 (t, 2H), 2.46 (s, 3H),1.78-1.87 (m, 1H), 1.61-1.67 (dd, 2H), 0.96 (s, 3H), 0.95 (3H). LRMS[M+H]=400.2

Example 1384-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl propylcarbonate

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) (1.0 equiv.) and triethyl amine (2 equiv.) indichloromethane (0.10 M) at 0° C. was added ethyl chloroformate (1.2equiv.). The resulting mixture was allowed to stir for 30 minutes at 0°C., after which it was diluted with water and dichloromethane. Thebiphasic layers were separated and the aqueous layer was washed twicewith dichloromethane. The combined organic layers were dried overanhydrous Na₂SO₄ and the volatiles were removed in vacuo. The resultingresidue was purified by a COMBIFLASH® system (ISCO) using 0-50% ethylacetate in hexanes to provide4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl ethylcarbonate as a solid. ¹H NMR (Acetone-d₆): δ 8.78 (s, 1H), 8.73 (s, 1H),8.28 (d, 1H), 7.43 (s, 1H), 7.33 (d, 2H), 7.10-7.17 (m, 3H), 6.64 (br,2H), 4.18 (t, 2H), 3.25 (t, 2H), 3.14 (t, 2H), 2.45 (s, 3H), 1.68-1.77(m, 2H), 0.97 (t, 3H). LRMS [M+H]=416.2

Example 139 ethyl5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)pentanoate

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) (1.0 equiv.) in dimethylformamide (0.10 M) 22° C. was added60% dispersion of sodium hydride in mineral oil (1.5 euiv.) and theresulting mixture was allowed to stir for 30 min. At this point, ethyl5-bromopentanoate (1.2 equiv.) was added to this mixture. The reactionmixture was then allowed to stir for 18 hours after which it was dilutedwith ethyl acetate and water. The biphasic layers were separated and theorganic layer was washed twice with water. The organic layer was driedover anhydrous Na₂SO₄ and the volatiles were removed in vacuo. Theresulting residue was purified by RP-HPLC using a 10-50% MeCN in watergradient. The resulting trifluoroacetate salt was then converted to thefree base form by utilizing a StratoSpheres™ PL-SO₃H SPE ion exchangeresin, delivering ethyl5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)pentanoateas a solid. ¹H NMR (Acetone-d6): δ 8.80 (s, 1H), 8.74 (s, 1H), 8.33 (d,1H), 7.47 (s, 1H), 7.24 (d, 1H), 7.17 (d, 2H), 6.85 (d, 2H), 4.10 (q,2H), 3.97 (t, 2H), 3.25 (t, 2H), 3.07 (t, 2), 2.50 (s, 3H), 2.37 (t,3H), 1.74-1.84 (m, 4H), 1.21 (t, 3H). LRMS [M+H]=458.2

Example 1402-(4-(cyclopentyloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(Cyclopentyloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 136, butusing bromocyclopentane. ¹H NMR (Acetone-d₆): δ 8.75 (d, 2H), 8.30 (d,1H), 7.45 (s, 1H), 7.20 (d, 1H), 7.14 (d, 2H), 6.79 (d, 2H), 4.73-4.81(m, 1H), 3.22 (t, 2H), 3.05 (t, 2H), 2.47 (s, 3H), 1.85-1.96 (m, 2H),1.70-1.79 (m, 4H), 1.56-1.64 (m, 2H). LRMS [M+H]=398.2

Example 1412-(4-(cyclobutylmethoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(Cyclobutylmethoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 136, butusing (bromomethyl)cyclobutane. ¹H NMR (Acetone-d₆): δ 8.79 (s, 1H),8.73 (s, 1H), 8.33 (d, 1H), 7.47 (s, 1H), 7.26 (d, 1H), 7.16 (d, 2H),6.82 (d, 2H), 3.90 (d, 2H), 3.23 (t, 2H), 3.06 (t, 2H), 2.68-2.79 (m,1H), 2.49 (s, 3H), 2.05-2.14 (m, 2H), 1.80-1.98 (m, 4H). LRMS[M+H]=398.2

Example 1428-methyl-2-(4-(2-morpholinoethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-(2-morpholinoethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 139, butusing 4-(2-bromoethyl)morpholine. ¹H NMR (Acetone-d₆): δ 8.78 (s, 1H),8.72 (s, 1H), 8.30 (d, 1H), 7.46 (s, 1H), 7.17-7.24 (m, 3H), 6.85 (d,2H), 4.08 (t, 2H), 3.56-3.62 (m, 4H), 3.45-3.53 (m, 2H), 3.24 (t, 2H),3.07 (t, 2H), 2.73 (t, 2H), 2.52-2.56 (m, 2H), 2.49 (s, 3H). LRMS[M+H]=443.2

Example 1432-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)-1-phenylethanone

2-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)-1-phenylethanonewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 139, butusing 2-bromo-1-phenylethanone. ¹H NMR (Acetone-d₆): δ 8.76 (s, 1H),8.71 (s, 1H), 8.27 (d, 1H), 8.06 (d, 2H), 7.67 (t, 1H), 7.57 (t, 2H),7.43 (s, 1H), 7.17 (d, 3H), 6.90 (d, 2H), 5.45 (s, 2H), 3.21 (t, 2H),3.06 (t, 2H), 2.45 (s, 3H). LRMS [M+H]=448.2

Example 1445-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)pentanoicacid

To a solution of ethyl5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)pentanoate(1.0 equiv.) (from Example 139) in ethanol (0.10 M) was added anhydroussodium hydroxide (2.0 equiv.) and the resulting mixture was allowed tostir at 80° C. for 2 hours. After cooling to ambient temperature, themixture was diluted with ethyl acetate and water. The biphasic layerswere separated and the aqueous layer was washed twice with ethylacetate. The combined organic layers were dried over anhydrous Na₂SO₄and the volatiles were removed in vacuo. The resulting residue waspurified by a COMBIFLASH® system (ISCO) using 0-10% methanol indichloromethane to furnish5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)pentanoicacid as a solid. ¹H NMR (Methanol-d₄): δ 8.61 (s, 1H), 8.57 (s, 1H),8.20 (d, 1H), 7.40 (s, 1H), 7.20 (d, 1H), 7.07 (d, 2H), 6.81 (d, 2H),3.93 (t, 2H), 3.18 (t, 2H), 3.00 (t, 2H), 2.48 (s, 3H), 2.25 (t, 2H),1.74-1.81 (m, 2H), 0.86-0.96 (m, 2H). LRMS [M+H]=430.2

Example 1452-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)ethanol

Step 1:2-(4-(2-(tert-butyldimethylsilyloxy)ethoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(2-(Tert-butyldimethylsilyloxy)ethoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 139, butusing (2-bromoethoxy)(tert-butyl)dimethylsilane.

Step 2:2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)ethanol

To a solution of2-(4-(2-(tert-butyldimethylsilyloxy)ethoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) (1.0 equiv.) in tetrahydrofuran (0.10 M) wasadded a 1.0 M solution of tetrabutylammonium fluoride (5 equiv.) in THFand the resulting mixture was allowed to stir at 22° C. for 2 hours. Atthis point, the mixture was diluted with ethyl acetate and water. Thebiphasic layers were separated and the aqueous layer was washed twicewith ethyl acetate. The combined organic layers were dried overanhydrous Na₂SO₄ and the volatiles were removed in vacuo. The resultingresidue was purified by a COMBIFLASH® system (ISCO) using 0-10% methanolin dichloromethane to furnish2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)ethanolas a solid. ¹H NMR (Acetone-d₆): δ 8.76 (s, 1H), 8.67 (s, 1H), 8.28 (d,1H), 7.40 (s, 1H), 7.15 (t, 3H), 6.84 (d, 2H), 6.54 (br, 2H), 4.00 (t,2H), 3.83 (t, 2H), 3.21 (t, 2H), 3.05 (t, 2H), 2.45 (s, 3H).

LRMS [M+H]=374.2

Example 1462-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)-N,N-dimethylacetamide

2-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)-N,N-dimethylacetamidewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) and following the procedure described for Example 139, butusing 2-bromo-N,N-dimethylacetamide. ¹H NMR (Acetone-d₆): δ 8.75 (s,1H), 8.70 (s, 1H), 8.28 (d, 1H), 7.40 (s, 1H), 7.18 (t, 3H), 6.87 (d,2H), 6.56 (br, 2H), 4.72 (s, 2H), 3.20 (t, 2H), 3.07 (s, 3H), 3.05 (t,2H), 2.87 (s, 3H), 2.45 (s, 3H). LRMS [M+H]=415.2

Example 1478-methyl-2-(2-methyl-4-(2-morpholinoethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(2-methyl-4-(2-morpholinoethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared following an analogous procedure to the preparationdescribed for Example 139, but using4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) and 4-(2-bromoethyl)morpholine. ¹H NMR (Acetone-d₆): δ8.73 (d, 2H), 8.26 (d, 1H), 7.44 (s, 1H), 7.17 (d, 1H), 7.05 (d, 1H),6.76 (s, 1H), 6.67 (d, 1), 4.04-4.08 (m, 3H), 3.60-3.62 (m, 4H), 3.30(s, 1H), 3.16 (t, 2H), 3.04 (t, 2H), 2.71 (t, 2H), 2.50-2.52 (m, 2H),2.47 (s, 3H), 2.28 (s, 3H). LRMS [M+H]=457.3

Example 1482-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol

Step 1:2-(4-(2-(2-(tert-butyldimethylsilyloxy)ethoxy)ethoxy)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(2-(2-(Tert-butyldimethylsilyloxy)ethoxy)ethoxy)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared following an analogous procedure to the preparationdescribed for Example 145/Step 1, but using4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) withtert-butyl(2-(2-chloroethoxy)ethoxy)dimethylsilane.

Step 2:2-(2-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol

2-(2-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanolwas prepared from2-(4-(2-(2-(tert-butyldimethylsilyloxy)ethoxy)ethoxy)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(from the previous step) following the procedures described for Example145/Step 2. ¹H NMR (Acetone-d₆): δ 8.74 (s, 1H), 8.69 (s, 1H), 8.27 (d,1H), 7.41 (s, 1H), 7.14 (d, 1H), 7.06 (d, 1H), 6.75 (s, 1H), 6.69 (d,1), 6.54 (br, 2H), 4.07 (t, 2H), 3.79 (t, 2H), 3.64 (t, 2H), 3.59 (t,2H), 3.16 (t, 2H), 3.03 (t, 2H), 2.45 (s, 3H), 2.29 (s, 3H). LRMS[M+H]=432.2

Example 149 diethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)propylphosphonate

Diethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)propylphosphonatewas prepared following an analogous procedure to the preparationdescribed for Example 139, but using4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) with diethyl 3-bromopropylphosphonate. ¹H NMR(Acetone-d₆): δ 9.52 (s, 1H), 9.47 (s, 1H), 9.03 (d, 1H), 8.21 (s, 1H),7.93 (d, 1H), 7.84 (d, 1H), 7.60 (br, 2H), 7.53 (s, 1), 7.45 (d, 1H),4.76-4.91 (m, 6H), 3.93 (t, 2H), 3.81 (t, 2H), 3.24 (s, 3H), 3.06 (s,3H), 2.76-2.86 (m, 2H), 2.61-2.72 (m, 2H), 2.07 (t, 6H). LRMS[M+H]=522.2

Example 1503-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)propylphosphonicacid

A 12 N solution of hycrochloric acid (0.10 M) was added to diethyl3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)propylphosphonate(from Example 149) and the resulting mixture was allowed to stir at 100°C. for 18 hours. At this point, hydrochloric acid was removed underreduced pressure and the resulting residue was purified by RP-HPLC usinga 10-50% MeCN in water gradient. The resulting trifluoroacetate salt wasthen converted to the free base form by the addition of a saturatedaqueous solution of sodium bicarbonate, followed by washing three timeswith ethyl acetate. The combined organic layers were dried withanhydrous Na₂SO₄, and the volatiles were removed in vacuo to deliver3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)propylphosphonicacid as a solid. ¹H NMR (Dimethylsulfoxide-d₆): δ 9.72 (br, 1H), 9.01(s, 1H), 8.96 (br, 1H), 8.85 (s, 1H), 8.54 (d, 1H), 7.54 (s, 1H), 7.42(d, 1H), 7.08 (d, 1), 6.74 (s, 1H), 6.66 (d, 1H), 3.95 (t, 2H), 3.14 (t,2H), 2.97 (t, 2H), 2.50 (s, 3H), 2.27 (s, 3H), 1.81-1.91 (m, 2H),1.56-1.67 (m, 2H). LRMS [M+H]=466.2

Example 1512-(4-butoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-Butoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared following an analogous procedure to the preparationdescribed for Example 139, but using4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) with 1-bromobutane. ¹H NMR (Acetone-d₆): δ 8.75 (s,1H), 8.71 (s, 1H), 8.28 (d, 1H), 7.43 (s, 1H), 7.15 (d, 1H), 7.07 (d,1H), 6.75 (s, 1H), 6.69 (d, 1H), 6.54 (br, 2H) 3.95 (t, 2H), 3.16 (t,2H), 3.04 (t, 2H), 2.47 (s, 3H), 2.30 (s, 3H), 1.69-1.77 (m, 2H),1.43-1.54 (m, 2H), 0.97 (t, 3H). LRMS [M+H]=400.2

Example 1522-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanol

Step 1: 4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl-3-methylphenol

4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol wasprepared following an analogous procedure to the preparation describedfor Example 145, but using2-(4-Methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine (fromExample 119).

Step 2:2-(4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanol

2-(4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanolwas prepared from4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol (fromthe previous step) following the procedures described for Example145/Steps 1 to 2. LRMS [M+H]=374.2

Example 1532-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanol

Step 1:4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol

4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol wasprepared following an analogous procedure to the preparation describedfor Example 50, but using2-(4-Methoxy-2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine (fromExample 119).

Step 2:2-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanol

2-(4-(2-(5-Aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanolwas prepared following the procedures described for Example 148/Steps 1to 2. LRMS [M+H]=418.2

Example 154 ethyl5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)pentanoate

Ethyl5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)pentanoatewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) following the procedure described for Example 139, butusing ethyl 5-bromopentanoate ¹H NMR (CDCl3): δ 8.64 (s, 1H), 8.27 (s,1H), 8.02 (d, 1H), 7.66 (s, 1H), 7.32 (d, 1H), 6.91 (d, 1H), 6.66 (s,1H), 6.63 (d, 1H), 4.13 (q, 2H), 3.93 (t, 2H), 3.14 (t, 2H), 2.99 (t,2H), 2.54 (s, 3H), 2.38 (t, 2H), 2.25 (s, 3H), 1.79-1.83 (m, 4H), 1.26(t, 3H). LRMS [M+H]=472.3

Example 1555-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)pentanoicacid

5-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)pentanoicacid was prepared from ethyl5-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)pentanoate(from the previous step) following the procedure described for Example144. ¹H NMR (CDCl3): δ 8.52 (s, 1H), 8.27 (s, 1H), 8.02 (d, 1H), 7.65(s, 1H), 7.32 (d, 1H), 6.86 (d, 1H), 6.72 (s, 1H), 6.63 (d, 1H), 3.95(t, 2H), 3.15 (t, 2H), 2.99 (t, 2H), 2.54 (s, 3H), 2.45 (t, 2H), 2.23(s, 3H), 1.79-1.83 (m, 4H).

LRMS [M+H]=444.2

Example 1562-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanol

2-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethanolwas prepared following the procedures described for Example 145/Steps 1to 2, but using4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50). ¹H NMR (Acetone-d₆): δ 8.76 (s, 1H), 8.69 (s, 1H),8.28 (d, 1H), 7.40 (s, 1H), 7.15 (d, 1H), 7.09 (d, 1H), 6.75 (s, 1H),6.68 (d, 1H), 6.57 (br, 2H), 4.00 (t, 2H), 3.79-3.88 (m, 2H), 3.17 (t,2H), 3.04 (t, 2H), 2.46 (s, 2H), 2.29 (s, 2H). LRMS [M+H]=388.5.

Example 1574-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl ethylcarbonate

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl ethylcarbonate was prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for Example 138, butusing ethyl carbonochloridate. LRMS [M+H]=402.2

Example 158 methyl4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)butanoate

Methyl4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)butanoatewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedure described for the preparation ofExample 139, but using methyl 4-bromobutanoate. ¹H NMR (Acetone-d₆): δ8.74 (s, 1H), 8.67 (s, 1H), 8.24 (d, 1H), 7.39 (s, 1H), 7.09-7.19 (m,3H), 6.82 (d, 2H), 6.53 (br, 2H), 3.97 (t, 2H), 3.60 (s, 3H), 3.19 (t,2H), 3.04 (t, 2H), 2.48 (t, 2H), 2.44 (s, 3H), 0.84-0.91 (m, 2H). LRMS[M+H]=430.2.

Example 1594-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)butanoicacid

4-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)butanoicacid was prepared from methyl4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenoxy)butanoate(from the previous step) following the procedure described for Example144. ¹H NMR (Acetone-d₆): δ 7.47 (s, 1H), 7.41 (s, 1H), 7.09 (d, 1H),6.21 (s, 1H), 6.18 (d, 1H), 5.82 (d, 2H), 5.52 (d, 2H), 2.66 (t, 2H),1.99 (t, 2H), 1.77 (t, 2H), 1.28 (s, 3H), 1.17 (t, 2H), 0.70-0.79 (m,2H). LRMS [M+H]=416.2.

Example 1604-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)butanoicacid

Step 1: methyl4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)butanoate

Methyl4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)butanoatewas prepared following the same procedure described for the preparationof Example 158, but using4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50).

Step 2:4-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)butanoicacid

4-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)butanoicacid was prepared from methyl4-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)butanoate(from the previous step) following the procedure described for Example144. ¹H NMR (Acetone-d₆): δ 8.38 (s, 1H), 8.24 (s, 1H), 7.90 (d, 1H),6.90 (s, 1H), 6.68 (d, 1H), 6.54-6.63 (m, 2H), 6.27 (d, 1H), 6.20 (d,1H), 3.40 (t, 2H), 2.62 (t, 2H), 2.47 (t, 2H), 1.99 (s, 3H), 1.80 (s,2H), 1.45 (t, 2H), 1.27-1.39 (m, 2H). LRMS [M+H]=430.2.

Example 1612-(4-(isopentyloxy)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(Isopentyloxy)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) following the procedure described for Example 136, butusing 1-bromo-3-methylbutane. ¹H NMR (Acetone-d₆): δ 8.75 (s, 1H), 8.72(s, 1H), 8.29 (d, 1H), 7.43 (s, 1H), 7.17 (D, 1H), 7.10 (d, 1H), 6.76(d, 1H), 6.68 (d, 1H), 6.56 (br, 2H), 4.00 (t, 2H), 3.17 (t, 2H), 3.07(t, 2H), 2.48 (s, 3H), 1.76-1.91 (m, 1H), 1.60-1.71 (m, 2H), 0.96 (s,6H).

LRMS [M+H]=414.2.

Example 1624-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl hexylcarbonate

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl hexylcarbonate was prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol (fromExample 170) following the procedures described for Example 138, butusing hexyl carbonochloridate. LRMS [M+H]=458.2.

Example 1632-(2,4,6-trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine

Step 1: 2-(mesitylethynyl)benzo[f][1,7]naphthyridin-5-amine

2-(Mesitylethynyl)benzo[f][1,7]naphthyridin-5-amine was prepared from3-chloro-5-(mesitylethynyl)picolinonitrile (Example 77/Step 1) andtert-butyl2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(commercially available) following the procedures described for Example45/Step 1.

Step 2: 2-(2,4,6-Trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine

2-(2,4,6-Trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine wasprepared from 2-(mesitylethynyl)benzo[f][1,7]naphthyridin-5-amine (fromthe previous step) following the procedures described for Example45/Step 2 to 3. ¹H NMR (Acetone-d₆): δ 8.80 (s, 2H), 8.38 (d, 1H), 7.60(d, 2H), 7.54 (d, 2H), 7.31 (t, 1H), 6.84 (s, 2H), 6.61 (br, 2H), 3.08(s, 2H), 2.30 (s, 6H), 2.23 (s, 3H). LRMS [M+H]=342.2.

Example 164(5-amino-2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

Step 1: methyl5-amino-2-(2,4-dimethylphenyl)ethynyl)benzo[f][1,7]naphthyridine-8-carboxylate

Methyl5-amino-2-((2,4-dimethylphenyl)ethynyl)benzo[f][1,7]naphthyridine-8-carboxylatewas prepared from 3-chloro-5-((2,4-dmethylphenyl)ethynyl)picolinonitrile (from Example 47/Step 3) and2-(tert-butoxycarbonylamino)-4-(methoxycarbonyl)phenylboronic acid (fromExample 85/Step 1) following the procedures described in Example 95/step1.

Step 2: methyl5-amino-2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridine-8-carboxylate

Methyl5-amino-2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridine-8-carboxylatewas prepared from methyl5-amino-2-((2,4-dimethylphenyl)ethynyl)benzo[f][1,7]naphthyridine-8-carboxylate(from the previous step) following the procedures described in Example44/Step 5.

Step 3:(5-amino-2-(2,4-dimethyl)phenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

(5-Amino-2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-8-yl)methanolwas prepared from methyl5-amino-2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridine-8-carboxylate(from the previous step) following the procedures described in Example95/Step 2. ¹H NMR (Acetone-d₆): δ 8.79 (s, 1H), 8.73 (s, 1H), 8.35 (d,1H), 7.61 (s, 1H), 7.34 (d, 1H), 7.08 (d, 1H), 6.97 (s, 1H), 6.91 (d,1H), 6.51 (br. 2H), 4.77 (s, 2H), 3.16-3.20 (m, 2H), 3.04-3.10 (m, 2H),2.28 (s, 3H), 2.25 (s, 3H). LRMS [M+H]=358.2.

Example 165 diethyl3-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)propylphosphonate

Diethyl3-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)propylphosphonatewas prepared following the procedure described for Example 139, butusing4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 156) and diethyl 3-(2-bromoethoxy)propylphosphonate. LRMS[M+H]=566.3.

Example 166 diethyl3-(2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)propylphosphonate

Diethyl3-(2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)propylphosphonatewas prepared from following the procedure described for Example 139, butusing4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 148) and diethyl3-(2-(2-bromoethoxy)ethoxy)propylphosphonate ¹H NMR (Acetone-d₆): δ 8.75(s, 1H), 8.70 (s, 1H), 8.26 (d, 1H), 7.42 (s, 1H), 7.16 (d, 1H), 7.09(d, 1H), 6.77 (s, 1H), 6.71 (d, 1H), 6.58 (br, 2H), 3.95-4.11 (m, 6H),3.76-3.80 (m, 2H), 3.63-3.67 (m, 2H), 3.55-3.58 (m, 2H), 3.57-3.51 (m,2H), 3.14-3.18 (m, 2H), 3.04-3.05 (m, 2H), 2.46 (s, 3H), 2.29 (s, 3H),1.71-1.87 (m, 4H), 1.22-1.29 (m, 8H). LRMS [M+H]=610.3.

Example 1674-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyldimethylsulfamate

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyldimethylsulfamate was prepared from4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenol(from Example 50) following the procedure described for Example 138, butusing dimethylsulfamoyl chloride. ¹H NMR (Acetone-d₆): δ 8.79 (s, 1H),8.72 (s, 1H), 8.28 (d, 1H), 7.42 (s, 1H), 7.27 (d, 1H), 7.17 (s, 1H),7.14 (t, 1H), 7.05-7.10 (d, 1H), 3.19-3.25 (m, 2H), 3.11-3.17 (m, 2H),2.92 (s, 6H), 2.46 (s, 3H), 2.37 (s, 3H). LRMS [M+H]=451.2.

Example 168(5-amino-2-(4-(dimethylamino)phenethyl)benzo[f][1,7]naphthyridin-8-yl)methanol

(5-Amino-2-(4-(dimethylamino)phenethyl)benzo[f][1,7]naphthyridin-8-yl)methanolwas prepared from tort-butyl5-((tert-butyldimethylsilyloxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(from Example 99/step 1) and 4-ethynyl-N,N-dimethylaniline (commerciallyavailable) following the procedures described in Example 45/Step 1 to 4followed by deprotection of TBS group as in Example 99/Step 3. ¹H NMR(Acetone-d₆): δ 8.78 (s, 1H), 8.73 (s, 1H), 8.35 (d, 1H), 7.61 (s, 1H),7.31-7.35 (d, 1H), 7.08 (d, 1H), 6.68 (d, 2H), 6.50 (br, 2H), 4.78 (s,2H), 4.34 (s, 1H), 3.16-3.20 (m, 2H), 3.03-3.10 (m, 2H), 2.83 (s, 3H),2.80 (s, 3H). LRMS [M+H]=373.2.

Example 1692-(4-(dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(Dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared following the procedures described for Example 45/Steps 1to 3, but using 4-ethynyl-N,N-dimethylaniline in step 1. ¹H NMR(Acetone-d₆) Free base: δ 8.60 (s, 1H), 8.55 (s, 1H), 8.15 (d, 1H), 7.28(s, 1H), 7.03 (d, 1H), 6.96 (d, 2H), 6.56 (d, 2H), 6.55 (br s, 2H), 3.05(t, 2H), 2.88 (t, 2H), 2.75 (s, 6H), 2.33 (s, 3H). LRMS [M+H]=357.2

Example 1704-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol

Step 1: 2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine wasprepared following the procedures described for Example 79/Steps 1 to 3,but using 1-ethynyl-4-methoxybenzene in Step 1.

Step 2:4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol wasprepared from2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine (fromthe previous step) following the procedure described for Example 50. ¹HNMR (Methanol-d₄): δ 8.59

Example 1711-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone

Step 1: 5-((4-acetylphenyl)ethynyl)-3-chloropicolinonitrile

To a solution of 1-(4-ethynylphenyl)ethanone (commercially available) (1eq) 3,5-dichloropicolinonitrile (1 eq),dichlorobis(triphenylphosphine)-palladium (II) (20 mol %), copper iodide(10 mol %) and DMF:Triethylamine (10:1) (0.13 M) was stirred at ambienttemperature overnight. The reaction mixture was then diluted with ethylacetate and sodium bicarbonate solution. The two phases were separated,and the aqueous phase was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over sodiumsulfate, and concentrated en vaccuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-100% ethylacetate in hexane and5-((4-acetylphenyl)ethynyl)-3-chloropicolinonitrile was isolated as ayellow solid

Step 2: 5-(4-acetylphenethyl)-3-chloropicolinonitrile

To a solution of 5-((4-acetylphenyl)ethynyl)-3-chloropicolinonitrile(from the previous step) (1 eq) in ethanol (0.1 M) was added PlatinumOxide (30 mol %). Hydrogen gas was introduced via a balloon, and thereaction was stirred for 0.5 hour. The mixture was filtered through apad of celite, washing with dichloromethane. The filtrate wasconcentrated en vaccuo and purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 0-100% ethyl acetate in hexane to give5-(4-acetylphenethyl)-3-chloropicolinonitrile as an off-white solid.

Step 3:1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone

To a solution of 5-(4-acetylphenethyl)-3-chloropicolinonitrile (from theprevious step) (1 eq) and tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(1.0 eq.), tetrakis(triphenyl-phosphine)palladium (10 mol %), and 2Naqueous sodium carbonate solution (2.0 eq.) in toluene/ethanol (1:1,0.09 M) was heated under microwave condition using a BIOTAGE INITIATOR2.0 at 150° C. for 20 minutes. After cooling to ambient temperature, thereaction mixture was diluted with ethanol/water. The insoluble solidswere filtered off, and the filtrate was concentrated en vaccuo to obtaina crude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane togive1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanoneas a yellow solid. ¹H NMR (Methanol-d₄) TFA Salt: δ 8.69 (d, 2H), 8.30(d, 1H), 7.80 (d, 2H), 7.38 (s, 1H), 7.36 (d, 1H), 7.28 (d, 2H), 3.25(t, 2H), 3.13 (t, 2H), 2.47 (s, 3H), 2.45 (s, 3H).

LRMS [M+H]=356.2

Example 1722-(4-((dimethylamino)methyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1:4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzaldehyde

4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzaldehydewas prepared from 4-ethynylbenzaldehyde (commercially available)following the procedures described for Example 171/Steps 1 to 3.

Step 2:2-(4-((dimethylamino)methyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzaldehyde(from the previous step) (1 eq), sodium acetate (3.5 eq) andN,N′-dimethyl amine hydrochloride (3.5 eq) dissolved in 1-2,dichloroethane (0.04 M) was heated at 80° C. for 2 hours in a sealedvial. After cooling to ambient temperature, the reaction mixture wasfurther cooled down to 0° C. and sodium tri-acetoxy borohydride (1.25eq) was added. The reaction mixture was stirred at room temperature forone hour. The mixture was diluted with ethyl acetate and water. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by preparative HPLC using 10-90% acetonitrile/water as thegradient and2-(4-((dimethylamino)methyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas isolated as a off-white powder as a TFA salt. ¹H NMR (Methanol-d₄)TFA Salt: δ 8.83 (s, 1H), 8.81 (s, 1H), 8.41 (d, 1H), 7.52 (s, 1H), 7.45(d, 1H), 7.43 (s, 1H), 7.40 (m, 3H), 4.29 (s, 2H), 3.30-3.24 (m, 4H),2.79 (s, 6H), 2.60 (s, 3H). LRMS [M+H]=371.2

Example 1732-(4-(1-(dimethylamino)ethyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(4-(1-(Dimethylamino)ethyl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-aminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) following the procedures described for Example172/Step 2. ¹H NMR (Methanol-d₄) TFA Salt: δ 8.84 (s, 1H), 8.79 (s, 1H),8.40 (d, 1H), 7.52 (s, 1H), 7.44-7.46 (m, 2H), 7.38-7.42 (m, 3H), 4.45(m, 1H), 3.31 (t, 2H), 3.19 (t, 2H), 2.83 (s, 3H), 2.66 (s, 3H), 2.56(s, 3H), 1.70 (d, 3H). LRMS [M+H]=385.2

Example 1741-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanoneoxime

A solution of1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) (1 eq), hydroxylamine hydrochloride (2 eq) and 1 dropof HOAc, dissolved in absolute ethanol (0.028M) was stirred at roomtemperature for 1.5 hours. The mixture was diluted with ethyl acetateand water. The two phases were separated, and the aqueous layer wasextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH® system (ISCO) using 10-80% ethyl acetate in hexane to give1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanoneoxime as a white solid. ¹H NMR (Methanol-d₄): δ 8.56 (s, 1H), 8.52 (s,1H), 8.12 (d, 1H), 7.45 (d, 2H), 7.31 (s, 1H), 7.12 (m, 3H), 4.51 (s,OH), 3.15 (t, 2H), 3.01 (t, 2H), 2.39 (s, 3H), 2.09 (s, 3H). LRMS[M+H]=371.2

Example 1758-methyl-2-(4-((methylamino)methyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-((methylamino)methyl)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzaldehyde(from Example 172/Step 1) and methylamine following the proceduresdescribed for Example 172, step 2. ¹H NMR (Acetone-d₆) TFA Salt: δ 8.95(s, 1H), 8.88 (s, 1H), 8.43 (d, 1H), 7.58 (s, 1H), 7.54 (d, 2H), 7.42(d, 1H), 7.37 (d, 2H), 4.30 (s, 2H), 3.32-3.37 (m, 4H), 2.75 (s, 3H),2.55 (s, 3H). LRMS [M+H]=357.2

Example 176(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzylamino)ethanol

A solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzaldehyde(from Example 172/Step 1) (1 eq), ethanol amine (8 eq) and 1 drop ofHOAc, dissolved in absolute ethanol (0.018M) was stirred at 80° C. for 2hours. The mixture was cooled down to 0° C. and NaBH₄ (3.5 eq) was addedand the reaction mixture was stirred for another one hour at roomtemperature. The mixture was diluted with ethyl acetate and water. Thetwo phases were separated, and the aqueous layer was extracted twicewith ethyl acetate. The combined organic layers were washed with brine,dried over anhydrous MgSO₄, and concentrated en vaccuo. The crudematerial was purified by Preparative HPLC on a 19×50 mm ATLANTIS® 10micron C18 (Waters Corp.) system using 10-90% Acetonitrile (0.035% TFA)in Water (0.05% TFA) to give a light yellow solid as a TFA salt. ¹H NMR(Acetone-d₆) TFA Salt: δ 8.82 (s, 1H), 8.75 (s, 1H), 8.30 (d, 1H), 7.44(m, 3H), 7.28 (d, 1H), 7.21 (d, 2H), 4.22 (s, 2H), 3.72 (t, 2H), 3.22(t, 2H), 3.09 (m, 2H), 3.07 (t, 2H), 3.01 (bs, OH), 2.41 (s, 3H), LRMS[M+H]=387.2

Example 1778-methyl-2-(4-(pyrrolidin-1-ylmethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-(pyrrolidin-1-ylmethyl)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzaldehyde(from Example 172/Step 1) and pyrrolidine following the proceduresdescribed for Example 172, step 2. ¹H NMR (Acetone-d₆) TFA Salt: δ 8.88(s, 1H), 8.82 (s, 1H), 8.82 (s, 1H), 8.43 (d, 1H), 8.38 (d, 1H), 7.58(s, 1H), 7.51 (m, 1H), 7.33 (d, 2H), 4.16 (s, 2H), 3.32-3.38 (m, 4H),2.55 (s, 3H), 2.20-2.32 (m, 4H), 1.90-1.99 (m, 4H). LRMS [M+H]=397.2

Example 1782-(3,4-dimethoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

2-(3,4-Dimethoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine wasprepared from 4-ethynyl-1,2-dimethoxybenzene (commercially available)following the procedures described for Example 45/Steps 1 to 3. ¹H NMR(Acetone-d₆): δ 8.64 (s, 1H), 8.56 (s, 1H), 8.14 (d, 1H), 7.29 (s, 1H),7.03 (d, 1H), 6.77 (s, 1H), 6.71 (s, 1H), 6.62 (d, 1H), 6.45 (bs, 2H),3.62 (s, 6H), 3.12 (t, 2H), 2.94 (t, 2H), 2.33 (s, 3H). LRMS [M+H]=374.2

Example 1792-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)ethanol

2-(1-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)ethanol(from Example 171) was prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanoneand ethanol amine (commercially available) following the proceduresdescribed for Example 176. ¹H NMR (Acetone-d₆) of TFA Salt: δ 8.78 (d,1H), 8.29 (d, 1H), 7.83 (s, 1H), 7.45 (m, 3H), 7.28 (m, 3H), 4.22 (m,1H), 3.52 (m, 2H), 3.23 (t, 2H), 3.09 (t, 2H), 2.85 (m, 1H), 2.65 (m,1H), 2.41 (s, 3H), 1.61 (d, 3H). LRMS [M+H]=401.2

Example 1801-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanol

1-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanol(from Example 171) was isolated as a side product during the reductiveamination as shown in Example 173. ¹H NMR (Acetone-d₆) of TFA Salt: δ8.90 (s, 1H), 8.88 (s, 1H), 8.42 (d, 1H), 7.57 (s, 1H), 7.43 (d, 1H),7.33 (d, 2H), 7.26 (d, 2H), 4.82 (q, 1H), 3.32 (t, 2H), 3.17 (t, 2H),3.01 2.55 (s, 3H), 1.41 (s, 3H). LRMS [M+H]=358.2

Example 1818-methyl-2-(4-(oxazol-5-yl)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-(oxazol-5-yl)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from 5-(4-ethynylphenyl)oxazole (commercially available)following the procedures described for Example 45/Steps 1 to 3. ¹H NMR(Acetone-d6) of TFA Salt: 8.69 (s, 1H), 8.59 (s, 1H), 8.16 (d, 1H), 8.04(s, 1H), 7.55 (m, 2H), 7.38 (s, 1H), 7.28 (m, 2H), 7.01 (m, 2H), 3.16(t, 2H), 3.07 (t, 2H), 2.33 (s, 3H). LRMS [M+H]=381.2

Example 1823-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)propanenitrile

A solution of1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) (1 eq), 3-aminopropane nitrile (commerciallyavailable) (2.5 eq) dissolved in absolute ethanol (0.014M) was stirredat 80° C. for 2 hours. The mixture was cooled to 0° C. and NaCNBH₃ (2eq) was added and the reaction mixture was stirred for another hour atroom temperature. The mixture was diluted with ethyl acetate andammonium chloride. The two phases were separated, and the aqueous layerwas extracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over anhydrous MgSO₄, and concentrated envaccuo. The crude material was purified by Preparative HPLC on a 19×50mm ATLANTIS® 10 micron C18 (Waters Corp.) system using 10-90%Acetonitrile (0.035% TFA) in Water (0.05% TFA) to give3-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)propanenitrileas a light yellow solid as a TFA salt. ¹H NMR (Acetone-d₆): δ 8.60 (s,1H), 8.59 (s, 1H), 8.11 (d, 1H), 7.29 (s, 1H), 7.16 (d, 2H), 7.09 (d,2H), 7.03 (d, 1H), 6.43 (bs, 2H), 3.65 (m, 1H), 3.12 (t, 2H), 2.99 (t,2H), 2.56 (m, 2H), 2.35 (m, 2H), 2.32 (s, 3H), 1.16 (d, 3H). LRMS[M+H]=410.2

Example 183(2R)-2-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)propan-1-ol

(2R)-2-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)propan-1-olwas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and (R)-2-aminopropan-1-ol (commercially available)following the procedures described for Example 182. ¹H NMR (Acetone-d₆):δ: 8.94 (m, 2H), 8.45 (m, 1H), 7.64 (d, 2H), 7.59 (s, 1H), 7.55 (br s,2H), 7.41 (m, 3H), 4.65 (m, 1H), 3.81 (m, 1H), 3.35 (t, 2H), 3.25 (t,2H), 2.56 (s, 3H), 1.73 (m, 3H), 1.29 (d, 3H), 1.23 (d, 3H). LRMS[M+H]=415.2

Example 1848-methyl-2-(4-(1-(piperazin-1-yl)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-(1-(piperazin-1-yl)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and piperazine (commercially available) following theprocedures described for Example 182. ¹H NMR (Methanol-d₄) TFA Salt: δ8.83 (s, 1H), 8.75 (s, 1H), 8.39 (d, 1H), 7.51 (s, 1H), 7.46 (d, 1H),7.26 (m, 4H), 3.62 (m, 1H), 3.25 (t, 2H), 3.12 (t, 2H), 2.80 (m, 4H),2.69 (m, 4H), 2.56 (s, 3H), 1.42 (d, 3H). LRMS [M+H]=426.2

Example 185((2S)-1-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)pyrrolidin-2-yl)methanol

((2S)-1-(1-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)pyrrolidin-2-yl)methanolwas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and (S)-pyrrolidin-2-ylmethanol (commerciallyavailable) following the procedures described for Example 182. ¹H NMR(Acetone-d₆) TFA Salt: δ 8.83 (s, 1H), 8.80 (s, 1H), 8.43 (d, 1H),7.36-7.53 (m, 6H), 4.68 (m, 1H), 3.69 (m, 2H), 3.19-3.21 (m, 4H), 2.55(m, 4H), 1.75-1.78 (m, 6H), 1.74 (d, 3H). LRMS [M+H]=441.2

Example 186N¹-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)-N²,N²-dimethylethane-1,2-diamine

N¹-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)-N²,N²-dimethylethane-1,2-diaminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and N′,N′-dimethylethane-1,2-diamine (commerciallyavailable) following the procedures described for Example 182. ¹H NMR(Acetone-d6) TFA Salt: δ 8.85 (m, 2H), 8.43 (d, 1H), 7.52 (s, 1H), 7.48(m, 2H), 7.40 (m, 2H), 6.69 (m, 1H), 4.39 (m, 1H), 3.42 (m, 2H),3.18-3.25 (m, 6H), 2.87 (s, 6H), 2.56 (s, 3H), 1.69 (d, 3H). LRMS[M+H]=428.2

Example 1873-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)propanoicacid

A solution of1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) (1 eq), 3-aminopropanoic acid (commerciallyavailable) (5 eq), triethylamine (3 eq) dissolved in absolute ethanol(0.042M) was stirred at 50° C. for 3 hours. The mixture was cooled to 0°C. and NaCNBH₃ (1 eq) was added and the reaction mixture was stirred foranother six hours at room temperature. Then another equivalent ofNaCNBH₃ was added and the reaction mixture was stirred at 50° C. foranother hour. After cooling to ambient temperature the reaction mixturewas diluted with ethyl acetate and saturated ammonium chloride. The twophases were separated, and the aqueous layer was extracted twice withethyl acetate. The combined organic layers were washed with brine, driedover anhydrous MgSO₄, and concentrated en vaccuo. The crude material waspurified by Preparative HPLC on a 19×50 mm ATLANTIS® 10 micron C18(Waters Corp.) system using 10-90% Acetonitrile (0.035% TFA) in Water(0.05% TFA) to give3-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)propanoicacid a white solid as a TFA salt. ¹H NMR (Methanol-d₄) TFA Salt: δ 8.74(s, 1H), 8.42 (d, 1H), 7.66 (m, 2H), 7.50 (m, 1H), 7.31 (d, 2H), 7.23(m, 2H), 4.24 (m, 1H), 3.21 (t, 2H), 3.14 (t, 2H), 2.75-3.10 (m, 2H),2.51 (t, 2H), 2.10 (s, 3H), 1.55 (d, 3H). LRMS [M+H]=429.2

Example 1888-methyl-2-(4-(1-(4-methylpiperazin-1-yl)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-(1-(4-methylpiperazin-1-yl)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and 1-methylpiperazine (commercially available)following the procedures described for Example 182. ¹H NMR (Acetone-d₆)TFA Salt: δ 8.84 (s, 1H), 8.80 (s, 1H), 8.41 (d, 1H), 7.52 (s, 1H),7.42-7.46 (m, 3H), 7.36-7.38 (m, 2H), 3.53 (m, 1H), 3.18 (m, 2H), 3.12(m, 2H), 2.92 (s, 2H), 2.66 (s, 2H), 2.56 (s, 2H), 2.16 (s, 3H), 1.99(m, 2H), 1.69 (d, 3H), 1.30 (s, 3H). LRMS [M+H]=440.2

Example 189N²-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)-N¹,N¹-dimethylpropane-1,2-diamine

N²-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)-N¹,N¹-dimethylpropane-1,2-diaminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and N¹,N¹-dimethylpropane-1,2-diamine (commerciallyavailable) following the procedures described for Example 182. ¹H NMR(Acetone-d6) TFA Salt: δ 8.83 (m, 2H), 8.40 (d, 1H), 7.46-7.51 (m, 3H),7.43 (m, 1H), 7.37 (d, 2H), 4.54 (m, 1H), 3.74 (m, 1H), 3.19 (m, 4H),2.90 (s, 3H), 2.77 (s, 3H), 2.55 (s, 3H), 2.41 (d, 2H), 1.66 (d, 3H),1.39 (d, 3H). LRMS [M+H]=442.2.

Example 1908-methyl-2-(4-(1-(2-(pyridin-4-yl)ethylamino)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine

8-Methyl-2-(4-(1-(2-(pyridin-4-yl)ethylamino)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-aminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and 2-(pyridin-4-yl)ethanamine (commerciallyavailable) following the procedures described for Example 182. ¹H NMR(Acetone-d₆) TFA Salt: δ 8.94 (m, 2H), 8.92 (d, 2H), 8.73 (s, 1H), 8.43(d, 1H), 7.60 (m, 2H), 7.40 (m, 2H), 7.16-7.26 (m, 3H), 4.55 (m, 1H),3.55 (m, 4H), 2.56 (m, 4H), 2.12 (s, 3H), 1.73 (d, 3H) LRMS [M+H]=462.2

Example 191

N¹-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)-N²,N²-diethylethane-1,2-diamine

N¹-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)-N²,N²-diethylethane-1,2-diaminewas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and N¹,N¹-diethylethane-1,2-diamine (commerciallyavailable) following the procedures described for Example 182. ¹H NMR(Acetone-d₆) TFA Salt: δ: 8.81 (s, 1H), 8.75 (s, 1H), 8.23 (d, 1H), 7.60(d, 2H), 7.39 (d, 2H), 7.28 (m, 2H), 4.51 (m, 1H), 3.82 (m, 1H), 3.62(m, 1H), 3.34 (m, 4H), 3.20 (t, 2H), 2.46 (s, 3H), 2.10 (m, 4H), 1.74(d, 3H), 1.34 (t, 6H). LRMS [M+H]=456.2

Example 1922-(4-(dimethylamino)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

Step 1: 4-iodo-N,N,3-trimethylaniline

To a solution of 4-iodo-3-methylaniline (commercially available) (1 eq),NaHCO₃ (2.5 eq), and iodomethane (2.5 eq), in DMF ((0.2M) was stirred atambient temperature overnight. The reaction mixture was then dilutedwith ethyl acetate and water. The two phases were separated, and theaqueous phase was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over sodium sulfate, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-80% ethyl acetatein hexane and 4-iodo-N,N,3-trimethylaniline was isolated as a yellowsolid.

Step 2: Syntheses of: N,N,3-trimethyl-4-((trimethylsilyl)ethynyl)aniline

To a solution of 4-iodo-N,N-3-trimethylaniline (from the previous step)(1 eq), ethynyltrimethylsilane (1.5 eq),dichlorobis(triphenylphosphine)-palladium (II) (20 mol %), copper iodide(20 mol %) and triethylamine (0.4 M) was stirred at ambient temperatureovernight. The reaction mixture was then diluted with ethyl acetate andammonium chloride solution. The two phases were separated, and theaqueous phase was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over sodium sulfate, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-100% ethyl acetatein hexane and N,N,3-trimethyl-4-((trimethylsilyl)ethynyl)aniline wasisolated as a yellow solid.

Step 3: 4-ethynyl-N,N,3-trimethylaniline

To a solution of N,N-3-trimethyl-4-((trimethylsilyl)ethynyl)aniline(from the previous step) (1 eq), K₂CO₃ (2.5 eq), in MeOH ((0.15M) wasstirred at ambient temperature for six hours. The solids were filteredout, and the liquid was concentrated en vaccuo. The crude material waspurified by flash chromatography on a COMBIFLASH® system (ISCO) using0-80% ethyl acetate in hexane and 4-ethynyl-N,N-3-trimethylaniline wasisolated as a yellow solid.

Step 4:3-chloro-5-((4-(dimethylamino)-2-methylphenyl)ethynyl)picolinonitrile

To a solution of 4-ethynyl-N,N-3-trimethylaniline (from the previousstep) (1 eq) 3,5-dichloropicolinonitrile (1.2 eq),dichlorobis(triphenylphosphine)-palladium (II) (10 mol %), copper iodide(10 mol %) and DMF: triethylamine (0.28 M) was stirred at ambienttemperature overnight. The reaction mixture was then diluted with ethylacetate and ammonium chloride solution. The two phases were separated,and the aqueous phase was extracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over sodiumsulfate, and concentrated en vaccuo. The crude material was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-100% ethylacetate in hexane and3-chloro-5-((4-(dimethylamino)-2-methylphenyl)ethynyl)picolinonitrilewas isolated as a off-yellow solid.

Step 5:2-((4-(dimethylamino)-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

A solution of tert-butyl5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate(1.3 eq.) and3-chloro-5-((4-(dimethylamino)-2-methylphenyl)ethynyl)picolinonitrile(from the previous step) (1.0 eq.),tetrakis(triphenyl-phosphine)palladium (10 mol %), and 2N aqueous sodiumcarbonate solution (2.0 eq.) in toluene/ethanol (2:1, 0.17 M) wasstirred at 100° C. overnight. After cooling to ambient temperature, thereaction mixture was diluted with methanol. The insoluble solids werefiltered off, and the filtrate was concentrated en vaccuo to obtain acrude residue. The crude material was purified by flash chromatographyon a COMBIFLASH® system (ISCO) using 0-80% ethyl acetate in hexane togive2-((4-(dimethylamino)-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amineas a yellow solid.

Step 6:2-(4-(dimethylamino)-2-methylyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a solution of2-(4-(dimethylamino)-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine(1 eq), (from the previous step) in ethyl acetate/ethanol (1:5, 0.035 M)was added 10% wt palladium on carbon (0.2 eq.). Hydrogen gas wasintroduced via a balloon, and the reaction was stirred for 3.5 hours.The mixture was filtered through a pad of celite, washing withdichloromethane. The filtrate was concentrated en vaccuo and purified bypreparative HPLC on a 19×50 mm ATLANTIS® 10 micron C18 (Waters Corp.)system using 10-90% Acetonitrile (0.035% TFA) in Water (0.05% TFA) togive2-(4-(dimethylamino)-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amineas a white solid as a TFA salt. ¹H NMR (Acetone-d6) TFA Salt: δ 8.81 (s,1H), 8.74 (s, 1H), 8.34 (d, 1H), 7.89 (s, 1H), 7.47 (m, 2H), 7.38 (m,2H), 3.34 (s, 6H), 3.32 (t, 2H), 3.28 (t, 2H), 2.57 (s, 3H), 2.34 (s,3H). LRMS [M+H]=371.2

Example 1931-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)pyrrolidine-3-carboxylicacid

1-(1-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)pyrrolidine-3-carboxylicacid was prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and pyrrolidine-3-carboxylic acid (commerciallyavailable) following the procedures described for Example 187, exceptthat in this case, acetic acid was used instead of triethylamine (30%).¹H NMR (Acetone-d₆) TFA Salt: δ 8.81 (s, 1H), 8.75 (s, 1H), 8.70 (s,1H), 8.28 (d, 1H), 7.59 (d, 2H), 7.37 (m, 3H), 4.46 (m, 1H), 4.21 (m1H), 3.45 (m, 2H), 3.32 (m, 2H), 3.21 (m, 2H), 3.17 (m, 2H), 2.27 (m,2H), 2.07 (s, 3H) 1.77 (d, 3H). LRMS [M+H]=455.2

Example 1944-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)phenol

4-(1-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethylamino)phenolwas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and 4-aminophenol following the procedures describedfor Example 187, except that in this case, acetic acid was used insteadof triethylamine (28%). ¹H NMR (Acetone-d₆) TFA Salt: δ 8.83 (s, 1H),8.73 (s, 1H), 8.33 (d, 1H), 7.44 (s, 1H), 7.40 (d, 2H), 7.36 (d, 1H),7.24 (d, 2H), 7.10 (d, 2H), 6.76 (d, 2H), 4.72 (m, 1H) 3.27 (t, 2H),3.12 (t, 2H), 2.50 (s, 3H), 2.06 (d, 3H). LRMS [M+H]=449.2

Example 195 1-(1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2yl)ethyl)phenyl)ethyl)pyrrolidin-3-ol

1-(1-(4-(2-(5-Amino-8-methylbenzo[f][1,7]naphthyridin-2yl)ethyl)phenyl)ethyl)pyrrolidin-3-olwas prepared from1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethanone(from Example 171) and pyrrolidin-3-ol following the proceduresdescribed for Example 187, except that in this case, acetic acid wasused instead of triethylamine (20%). ¹H NMR (Acetone-d₆) TFA Salt: δ8.83 (s, 1H), 8.76 (s, 1H), 8.72 (s, 1H), 8.29 (d, 1H), 7.57 (s, 1H),7.42 (s, 1H), 7.33-7.38 (m, 3H), 4.41 (m, 2H), 3.77 (m, 2H), 3.33 (t,2H), 3.21 (t, 2H), 3.19 (m, 2H), 3.10 (m, 2H), 2.10 (s, 3H), 1.75 (d,3H). LRMS [M+H]=427.2

Example 1962-(4-(2-aminopropan-2-yl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzonitrile(from Example 64, step 1) (1 eq), dissolved in dry THF (0.029M) wasadded very slowly methyl magnesium bromide (6 eq) and the reactionmixture was stirred at room temperature for half hour. Then was added tothe reaction flask titanium tetra-isopropoxide (3 eq) over ten minutes.The reaction mixture was refluxed for 16 hours. After cooling to ambienttemperature the reaction mixture was diluted with ethyl acetate andsaturated ammonium chloride. The two phases were separated, and theaqueous layer was extracted twice with ethyl acetate. The combinedorganic layers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vaccuo. The crude material was purified by PreparativeHPLC on a 19×50 mm ATLANTIS® 10 micron C18 (Waters Corp.) system using10-90% Acetonitrile (0.035% TFA) in Water (0.05% TFA) to give2-(4-(2-aminopropan-2-yl)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amineas a white solid of TFA salt. ¹H NMR (Methanol-d₄) TFA Salt δ: 9.01 (s,2H), 8.92 (s, 1H), 8.42 (s, 1H), 7.65 (d, 2H), 7.56 (s, 1H), 7.39 (m,2H), 3.19 (m, 4H), 2.54 (s, 3H), 1.82 (6H). LRMS [M+H]=371.2.

Example 197 N-(2-acetamidoethyl)-4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamide

To a solution of4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoylchloride (Example 116/Step 2) and triethylamine (2.5 eq.) in ether (0.05M) was added N-(2-aminoethyl)acetamide (5.0 eq.). The reaction mixturewas stirred for overnight. Then the reaction mixture was diluted withethyl acetate and water. The two phases were separated, and the aqueouslayer was extracted twice with ethyl acetate. The combined organiclayers were washed with brine, dried over anhydrous MgSO₄, andconcentrated en vaccuo. The crude material was purified by flashchromatography on a COMBIFLASH® system (ISCO) using 0-80% ethyl acetatein hexane to giveN-(2-acetamidoethyl)-4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamideas a white solid. ¹H NMR (CDCl₃): δ 8.61 (s, 1H), 8.38 (s, 1H), 8.07 (d,1H), 7.65 (s, 1H), 7.51-7.56 (m, 2H), 7.10-7.16 (m, 2H), 6.25 (br, 2H),3.50-3.59 (m, 4H), 3.08-3.16 (m, 4H), 2.62 (s, 3H), 2.52 (s, 3H), 2.35(s, 3H). LRMS [M+H]=455.2.

Other compounds for carrying out the present invention include:2-methylbenzo[f][1,7]naphthyridin-5-amine;2-propylbenzo[f][1,7]naphthyridin-5-amine;2-(3-methoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-phenethylbenzo[f][1,7]naphthyridin-5-amine;methyl-5-aminobenzo[f][1,7]naphthyridine-3-carboxylate;(5-aminobenzo[f][1,7]naphthyridin-3-yl)methanol;2-(2-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(3-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-methylphenethyl)benzo[f][1,7]naphthyridin-5-amine,8-methyl-2-(2-(naphthalen-1-yl)ethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(2-(naphthalen-2-yl)ethyl)benzo[f][1,7]naphthyridin-5-amine;4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoic acid;3-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzoic acid;2-(3-chlorophenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(2-chlorophenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;(3-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)methanol;2-(4-chlorophenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-butylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-butylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-propylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(trifluoromethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(2,5-dimethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-propylphenethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(2,4,5-trimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(2,5-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-isopropylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-heptylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-isobutoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-((2-methoxyethoxy)methoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(2-phenoxyethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(4-phenylbutoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(alkyloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(3-phenylpropoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(heptan-4-yloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(4-methylpent-3-enyloxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(2-cyclohexylethoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-isopropoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-(3,3-dimethylbutoxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-(2-cyclopropylethyl)-2-(4-(dimethylamino)phenethyl)benzo[f][1,7]naphthyridin-5-amine;8-(2-cyclopropylethyl)-2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine;N-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)acetamide;N-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)-4-methylbenzenesulfonamide;3-methyl-9-p-tolyl-9,10-dihydrobenzo[f]furo[2,3-b][1,7]naphthyridin-6-amine;4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzonitrile;4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-(2-aminoethyl)-3-methylbenzamide;8-methyl-2-(2-methyl-4-(1H-tetrazol-5-yl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;methyl2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamido)-4-methylpentanoate;methyl2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamido)acetate;2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamido)-4-methylpentanoicacid;2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzamido)aceticacid; 6-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)hexan-1-ol;7-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)heptanoic acid;11-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)undecan-1-ol; ethyl2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)acetate;2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)aceticacid;3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)propanoicacid;6-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)hexanoicacid;8-methyl-2-(2-methyl-4-(methylthio)phenethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(methylsulfonyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(hexyloxy)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-phenethoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(pentyloxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(4-methylpentyloxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(2-fluorophenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(3-fluorophenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-fluorophenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(2-(thiophen-3-yl)ethyl)benzo[f][1,7]naphthyridin-5-amine;(5-aminobenzo[f][1,7]naphthyridin-2-yl)methanol;2-(3,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(3,4-dimethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(3,5-dimethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(2-(benzo furan-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(2-nitro ethyl)benzo[f][1,7]naphthyridin-5-amine;2-(aminomethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;N²,8-dimethylbenzo[f][1,7]naphthyridine-2,5-diamine;2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)-1-phenylethanol;2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)-1-(4-methoxyphenyl)ethanol;2-(biphenyl-2-yl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(2-(2,6-dimethylpyridin-3-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(2-(5-methoxypyridin-2-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)propanoicacid;5-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-4-methylpyridin-2(1H)-one;6-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)pyridin-3-ol;8-methyl-2-(4-(trifluoromethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(2-(2,3-dihydro-1H-inden-5-yl)ethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(2-(2,3-dihydro-1H-inden-5-yl)ethyl)benzo[f][1,7]naphthyridin-5-amine;(E)-3-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)acrylicacid;(E)-8-(2-cyclopropylvinyl)-2-phenethylbenzo[f][1,7]naphthyridin-5-amine;8-pentylbenzo[f][1,7]naphthyridin-5-amine;(E)-8-(2-cyclopropylvinyl)benzo[f][1,7]naphthyridin-5-amine;8-(2-cyclopropylethyl)-2-phenethylbenzo[f][1,7]naphthyridin-5-amine;3-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)phenol;2-(2-methoxyphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-ethylphenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-ethylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(4-(dimethylamino)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(piperidin-1-yl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-tert-butylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(4-(piperidin-1-yl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;2-(3,5-dimethoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;8-methyl-2-(2-(trifluoromethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-N-hydroxybenzimidamide;4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)benzonitrile;8-methyl-2-(4-(1-morpholinoethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-aminophenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine;1-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)guanidine;8-methyl-2-(4-(1-(phenethylamino)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)acetonitrile;2-(4-(piperidin-1-ylmethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;1-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)benzyl)piperidin-4-ol;2-(4-(aminomethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-((ethylamino)methyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;2-(4-(2-aminopropan-2-yl)phenethyl)benzo[f][1,7]naphthyridin-5-amine;1-(1-(4-(2-(5-aminobenzo[f][1,7]naphthyridin-2-yl)ethyl)phenyl)ethyl)pyrrolidine-3-carboxylicacid, and 8-methyl-2-(4-(1-(phenylamino)ethyl)phenethyl)benzo[f][1,7]naphthyridin-5-amine.

Example (II) Development of a Homogeneous Suspension of Example 47

Formulations of small molecule immune potentiators (SMIPs) wereevaluated for stability and activity as vaccine adjuvants orimmunomodulatory agents against bacterial and viral infections.

Example 47 is a highly crystalline drug substance with very goodchemical stability. Due to its extremely low aqueous solubility acrossfull pH range (<0.5 μg/ml) and its high hydrophobic core structure,formulation with Alum (the only vaccine adjuvant licensed in the U.S.)is challenging. However, TLR7 agonist-Alum based vaccines could provideadvantages, for example, induction of Th1 polarized response, andenhanced Serum Bactericidal Activity (SBA) and breath of coverage (e.g.,MenB).

This example describes the development of a stable and reproduciblehomogeneous suspension of Example 47 as a vehicle to deliver themolecule in vivo, with particle sizes in the micron range.

Evaluation of Surfactants, Suspension Agents and Homogenization Methods

A number of homogenization methods were evaluated, including vortexing,sonication, and mortar and pestle method.

For vortexing, Example 47 was suspended at 2.5 mg/mL in 0.1% Tween 80and 0.5% carboxymethyl cellulose (CMC), followed by vortexing. Theaverage particle size was ˜100 μm.

For sonication, Example 47 was suspended at 2.5 mg/mL in 0.1% Tween 80and 0.5% CMC followed by vortexing. The mixture was then subject toprobe sonication for 5 minutes (with ice at 3 W, maximum power based onvial size). Finally, the mixture was homogenized by a handheldhomogenizer for a few minutes. The average particle size was ˜80 μm.

In mortar and pestle method, 10 mg of Example 47 was ground for 5minutes using mortar and pestle, then suspended in 0.5% Tween 80. Twoparticle size peaks were observed with particle sizes of 0.1 μm and ˜8μm. Size range for the larger peak was 1-100 μm.

A number of surfactants and suspension agents were also evaluated. Thematrix of surfactants and suspending agents, and the corresponding sizesdistributions of the suspension particles are shown in the Table 1 below(D50, D90 (μm)).

TABLE 1 0.5% 0.5% 0.1% 0.5% Plutonic Sodium Tween 80 Tween 80 F-68Lauryl Sulfate 0.5% CMC 76.2, 144.7 75.8, 149.1 49.1, 104.4 28.0, 53.30.5% HPMC 25.8, 53.5 27.5, 49.7 19.4, 33.5 26.8, 59.3 0.5% HPC 43.6,68.0 35.4, 53.5 35.8, 54.9 38.9, 53.1

In each experiment, 10 mg of Example 47 was mixed with 2 mL surfactantand 2 mL suspending agent. The mixtures were vortexed for 5 minutes, andprobe sonicated at 3 W and homogenized for 2-4 minutes. The sizedistributions of the samples were measured using Mastersizer.

All samples were observed to settle quickly even during the course ofprobe sonication. No visual size reduction was observed aftersonication. Some size reduction was noticed visually afterhomogenization.

High Pressure Homogenization

Evaluation of a number of surfactants and suspension agents identified astable homogeneous suspension after high pressure homogenization ofExample 47 with the surfactant Tween-80 and the suspension agentcarboxymethyl cellulose (CMC). Homogenization was carried out at highpressure on ice for up to 30 minutes.

1% CMC (carboxymethyl cellulose, sodium salt; sigma 419273, M5013) stocksolution in milliQ water, and 1% Tween 80 solution in MilliQ water wereprepared. Crystalline Example 47 (80 mg) was mixed with equal volumes(10 mL each) of 1% CMC and 1% Tween 80 to a final compound concentrationof 4 mg/ml (in 0.5% CMC and 0.5% Tween 80). The mixture was first vortexthoroughly and homogenized for 1 min at 24000 rpm using IKA T-25homogenizer, then processed through a high pressure homogenizer (AvestinEmulsifex-C3) at high pressure (15,000-20,000 psi) on ice for 20-30minutes. The size distributions of samples were measured. Thehomogenization procedure was considered complete once the sizedistribution of at least about 50% of the suspension particles had adiameter (D50) of about 2 micrometers or less, and at least about 90% ofthe suspension particles had a diameter (D90) of about 10 micrometers orless, preferably about 5 micrometer or less. The homogenized suspensionwas stored at 4° C.

Homogeneous suspensions of Example 47 were independently reproduced attwo separate locations (“Suspension S” and “Suspension C”) using thesame protocols. The homogeneous suspensions of Example 47 (created underhigh pressure) had particle sizes in the μm range, as measured by aHoriba particle sizer. The particle size distributions of the twosuspensions are very similar. Further, the suspensions showed goodstability over time—size distribution of the particles, as representedby D50 and D90, had not changed significantly after 30 days and 56 days.Table 2 shows that Suspension S was stable over 4 weeks at 4° C.(measured by Beckman coulter particle sizer).

TABLE 2 Example 47 suspension was stable over 4 weeks at 4° C. Timeafter preparation D50 (μm) D90 (μm) Day 1 1.25 3.37 Day 30 1.46 5.92 Day56 1.45 7.3

Table 3 shows that the homogeneous suspension of Example 47 was stableover 12 months at 4° C., as measured by size distribution.

TABLE 3 Example 47 suspension was stable over 12 months at 4° C. Timeafter preparation D50 (μm) D90 (μm) Mean (μm) 0 0.5 (0.5243) 0.9(0.9236) 0.6 (0.5828) 1 0.6 (0.5860) 1.1 (1.1114) 0.7 (0.6676) 6 0.7(0.6830) 1.4 (1.3556) 0.8 (0.7882) 9 0.6 (0.6493) 1.3 (1.2869) 0.8(0.7540) 12 0.7 (0.6566) 1.4 (1.3736) 0.8 (0.7821)

Example (III) Formulation of Example 47 Homogeneous Suspension with AlumBased Vaccines: Effect on Antigen Adsorption

Meningococcus B

Giuliani et al. (Proc. Natl. Acad. Sci. USA 103:10834-10839 (2006))discloses a vaccine for serogroup B meningococcus (“MenB”) made fromthree separate polypeptides (“5CVMB”). These polypeptides can adsorb toaluminium hydroxide (“Al—H”), and SDS-PAGE is used to check if thisadsorption occurs.

In this study, effects of formulating different doses of Example 47suspension with the MenB antigens (“5CVMB”) plus Al—H (3 mg/ml)(“Alum/3MenB”) were evaluated. As shown in FIGS. 1A and 1B, when no (0μg/dose) suspension was added, the three antigens tested were completelyadsorbed on to Al—H. As the dose of the suspension was increased, smallamount of antigens were de-adsorbed from Al—H in a time-dependentmanner. FIG. 1B is a table showing the percentage of antigen adsorptiononto aluminium hydroxide. Although some dose-dependant desorption of theMenB antigens was observed after formulation with the Example 47suspension, each of the three antigen remained >60% adsorbed after 2hours at 4° C.

Extraintestinal Pathogenic E. coli (“ExPEC”)

In this study, effects of formulating different doses of Example 47 withExPEC antigen pK1-3526 plus Al—H were evaluated. Example 47 suspensionswere stored at 4° C. for 16 hours, and then added to various pK1-3526formulations. As shown in FIG. 2, pK1-3526 antigen remained adsorbedwith high efficiency (over 90%) and changes in antigen integrity upondesorption were not detected.

Studies have also been conducted with respiratory syncytial virus (RSV)antigens with similar results.

The MenB, ExPEC and RSV studies show that the combination of the Example47 suspension with antigen/Al—H formulations had no impact on adsorptionof MenB, ExPEC and RSV antigens. Some desorption of MenB antigens fromAl—H was observed with high doses of Example 47 suspension, but thiseffect was time dependent and was reduced when the suspension was mixedwith the vaccine just prior to immunization.

Example (IV) Pharmacokinetic and Pharmacodynamic Parameters of theHomogeneous Suspension of Example 47

In this study, pharmacokinetic (PK) and pharmacodynamic (PD) parametersof the homogeneous suspension of Example 47 plus Alum/3MenB werestudied, and were found to be similar to Example 47 prepared as anon-homogeneous suspension in DMSO/saline (FIGS. 3A, 3B and 4). A slightincrease in detectable systemic levels of Example 47 formulated in thehomogeneous suspension was identified (FIG. 3A). PK studies demonstratelocal exposure of Example 47 in both DMSO (non-homogeneous) andhomogeneous suspensions, but low systemic exposure. Homogeneoussuspension reduced variability in the local muscle exposure and improvesexposure in the local draining lymph node (FIGS. 3B and 3C).

FIG. 4 shows the pharmacodynamic comparison of non-homogeneous/DMSOsuspension and homogeneous suspension of Example 47 plus Alum/3MenB. Thecytokine release profiles of non-homogeneous/DMSO suspension andhomogeneous suspension of Example 47 were similar. Cytokine releaseswere minimal as compared to cytokine release induced by the adjuvantR848 after exposure of homogeneous suspensions or DMSO formulatedExample 47.

Example (V) Efficacy of Homogeneous Suspension of Example 47 in In VivoMenB Model

1. Example 47 Homogeneous Suspension Enhanced Immune Responses toNeisseria meningitis

In this study, the in vivo efficacy of Example 47, delivered ashomogeneous suspension at doses ranging between 2 and 100 μg/mouse, wasevaluated using a MenB mouse model. The evaluation was carried out withthe three MenB polypeptides (“5CVMB”) either adsorbed on Al—H(“Alum/3MenB”) or delivered with MF59 (“MF59/3MenB”). The McnB antigenswere tested for in vivo immunogenic potency using a serum bactericidalantibody (SBA) assay. FIG. 5A shows bactericial titers against strainNZ98 of sera obtained after immunization with 5CVMB combined with (a)Al—H alone, (b) Al—H+2 μg Example 47, (c) Al—H+10 μg Example 47, (c)Al—H+25 μg Example 47, (d) Al—H+100 μg Example 47 or (e) Al—H and MenBouter membrane vesicles. FIG. 5B shows bactericial titers against strainNZ98 of sera obtained after immunization with 5CVMB combined with (a)MF59 alone, (b) MF59+2 μg Example 47, (c) MF59+10 μg Example 47, (c)MF59+25 μg Example 47, (d) MF59+100 μg Example 47 or (e) MF59 and 25 μgCpG1826.

The Alum/3MenB formulations (FIG. 5A) were prepared at 75% volume, andExample 47 homogeneous suspension (4 mg/mL) was added at doses 0, 2, 10,25 and 100 μg, respectively, within 2 hours prior to immunization. CD1mice were immunized intramuscularly on days 0 and 14 with 100 μLimmunization volume (50 μL/leg) containing 10 μg of each MenB antigenand the specified Example 47 homogenous suspension doses. Mice were bledretro-orbitally on day 28 and SBA titers were measured using Alamar Blueassay. Alum/3MenB/OMV was used as a benchmark.

The MF59/3MenB formulations (FIG. 5B) were prepared by adding the MenBantigens (5CVMB) (100 μg/mL each) and MF59 to the Example 47 homogeneoussuspension (4 mg/mL) at concentrations of 0, 20, 100, 250 and 1,000μg/mL, respectively. CD1 mice were immunized intramuscularly on days 0and 14 with 100 uL immunization volume (50 μL/1 eg) containing 10 μg ofeach MenB antigen and the specified Example 47 homogeneous suspensiondoses. Mice were bled retro-orbitally on day 28 and SBA titers weremeasured using Alamar Blue assay. MF59/3MenB+25 μg CpG1826 was used as abenchmark.

As shown in FIG. 5A, Alum/3MenB plus Example 47 homogeneous suspensionat 10 μg, 25 μg and 100 μg gave titers that were significantly enhancedrelative to the SBA titers seen with Alum/3MenB alone, and SBA titersthat were comparable to, or better than, SBA titers obtained usingAlum/3MenB/OMV. Similar results were obtained using MF59/3MenBformulations.

2. Homogeneous Suspension of Example 47 Improved Strain CoverageCompared to Al—H Alone

Strain coverage of sera obtained after intramuscular immunization ofmice with 5CVMB combined with (a) Al—H alone, (b) Al—H+100 μg Example 47(DMSO), (c) Al—H+25 μg Example 47 homogeneous suspension, (c) Al—H+100μg Example 47 homogenous suspension and (d) Al—H and MenB outer membranevesicles, and intraperitoneal immunization of mice with 5CVMB combinedwith (e) Al—H and MenB outer membrane vesicles, was evaluated.Alum/3MenB/OMV was used as a benchmark. Efficacy readout is expressed asthe % of strains reaching a defined threshold of killing in the SBAassay in the MenB mouse model.

Table 4 shows the sixteen (16) different strains of Neisseriameningitides that were used in these studies. The 16 strains wereselected to represent (1) specific target for only one of the antigeniccomponents of 5CVMB; and (2) historically low or negative SBA titerswith Alum/3MenB. SBA titers of >1084 was used as a cut off to indicatecoverage against a particular strain.

TABLE 4 cpx ST ET YEAR TYPING 741 961 NZ98/254 41/44 42 lin3 1998 NZB:4:P1.4 1.10/++ − H44/76 32 32 ET5 1976 N B:15:P1.7, 16   1.1/+++ −M01-240 185 11 11 ET37 2001 UK B:2a:P1.5, 10   1.9/+++ − M2937 35 newother 1996 USA B:4, 7:P1.23, 14   1.7/+++ − 2996 8 540 A4 1975 UKB:2b:P1.5, 2 2.1/+ + 961-5945 8 153 A4 1996 AUS B:2b:P1.21, 16  2.1/++ +B3937 18 new other 1995 D B:22:P1.16   2.2/+++ + M2552 — 103 other 1996USA B:NT:NT 2.10/+  − M4407 41/44 new lin.3 1996 USA B:NT:P1.15  2.4/++− M0579 41/44 43 lin.3 1993 USA B:NT:P1.5, 2 2.5/+ − M986 11 11 ET371963 USA B:2a:P1.5, 2 2.7/− + 5/99 8 1349 A4 1999 N B:2b:P1.5, 2 2.8/+ +M4458 22 new other 1998 USA B:NT:P1.3 2.10/+  + NGP165 11 11 ET37 1974 NB:NT:P1.2   3.2/+++ + M01-0240364 11 11 ET37 2001 UK B:2a:P1.5, 23.4/+ + M3369 — 1576 other 1997 USA B:10:P1.19, 15  3.4/++ −

CD1 mice were immunized following an immunization schedule of 1 and 14days. At each immunization, 0.1 ml dose of each formulation wasdelivered via intramuscular route of 0.05 ml to each of two legs. Themice were bled at 28 days and sera analyzed using SBA assay. Anadditional control group was included consisting of 0.2 ml Alum/3MenBplus OMV delivered via intraperitoneal route at 1 and 14 days. Twostudies, with comparable immunization schemes, were conducted (“MenBStudy 1” and “MenB Study 2”), with both giving comparable data.

Table 5 shows SBA titers against strain NZ98/254 in two separatestudies.

TABLE 5 Group Antigens Route NZ Study 1: MenB + Example 47 1 Alum/PBS IM<16 2 Alum/3MenB IM 128 3 Alum/3MenB + 100 μg Ex. 47 (DMSO) IM 8192 4Alum/3 MenB + 25 μg IM 2048 Ex. 47 homo. suspension 5 Alum/3 MenB + 100μg IM 1024 Ex. 47 homo. suspension 6 Alum/3MenB + OMV IM 128 7Alum/3MenB + OMV IP 16384 Study 2: MenB + Example 47 1 Alum/PBS IM <16 2Alum/3MenB IM 128 3 Alum/3MenB + 100 μg Ex. 47 (DMSO) IM 4096 4 Alum/3MenB + 25 μg IM 4096 Ex. 47 homo. suspension 5 Alum/3 MenB + 100 μg IM2048 Ex. 47 homo. suspension 6 Alum/3MenB + OMV IM 1024 7 Alum/3MenB +OMV IP 8192

As shown in FIG. 6, Alum/3MenB formulated with Example 47 homogenoussuspension (intramuscular, i.m.) was able to cover 75% of strains usedin this experiment, while Alum/3MenB alone was able to cover only 19%.Alum/3MenB/OMV was able to cover ˜50% of strains when formulated in Al—Hand delivered i.m., and 80% of strains when via deliveredintraperitoneal (i.p.) route. The dose-dependent effect was not evident.Thus, Alum/3MenB plus Example 47 homogeneous suspension at 25 μg and 100μg improved strain coverage compared to Alum/3MenB alone.

FIGS. 7A-7C shows the IgG1/IgG2a ratios in the MenB mouse model in vivo.Total IgG titres were calculated by ELISA. FIG. 7A shows the IgG1,IgG2a, IgG2b and IgG3 titres for GNA2132-GNA1030. FIG. 7B shows theIgG1, IgG2a, IgG2b and IgG3 titres for GNA2091-GNA1870. FIG. 7C showsthe IgG1, IgG2a, IgG2b and IgG3 titres for NadA. Table 6 summarizes theresults from an ELISA assay that demonstrated the ability of Example 47suspension to induce IgG2a when added to Alum/3MenB. Example 47 enhancedIgG2a isotype switching (decreased ratio of IgG1/IgG2a) when formulatedwith Alum/3MenB (the lower the number, the bigger the IgG2a foldincrease was induced by Example 47 suspension). IgG2a is considered as asurrogate of Th1 response is desirable to elicit.

TABLE 6 IgG1/IgG2a ratios GNA2132- GNA2091- GNA1030 GNA1870 NadAAlum/3MenB 923 15 308 Alum/3MenB + 100 μg Example 47 1 2 7 Alum/3MenB +OMV 36 6 61

This Example demonstrates that the Example 47 homogenous suspension cansignificantly enhance immune responses to Alum/3MenB, with SBA titerscomparable to, or better than, Alum/3McnB/OMV at 10 μg/dose, 25 μg/dose,or 100 μg/dose of Example 47 suspension. Further, Example 47 suspensioncan increase breadth of the immune response, as shown by an increase inthe breadth of protection to various Neisseria meningitis strains.

Significantly, when non-homogeneous Example 47 suspension (prepared inDMSO) were used in conjunction with aluminium-based vaccines andMF59-based vaccines, the non-homogeneous suspension resulted in lowerSBA titers when compared to the effects of homogeneous suspension at thesame does. In one set of studies using the MenB mouse model, SBA titersupon addition of 10 μg/dose of non-homogeneous Example 47 suspension wascomparable to that of MF59/3MenB alone (FIG. 8). In another set ofstudies, non-homogeneous Example 47 suspension showed only a borderlineadjuvant effect. Therefore, the homogeneous suspensions described inthis invention not only reduced the high degree of variability betweenthe replicate animals and increase bioavailability of the molecules toimmune cells post immunization, but also provide immuno-stimulatoryeffect at lower doses (e.g., 10 μg/dose or less).

Example (VI) Efficacy of Homogeneous Suspension of Example 47 in In VivoRSV Model

In this example, the in vivo efficacy of Example 47, delivered ashomogeneous suspension, was evaluated using an RSV model.

A. Study 1

In one study, RSV F-subunit antigen was adsorbed on Al—H at a targetconcentration of 50 μg/mL or 10 μg/mL in 10 μM Histidine buffer. Thesealum formulations were prepared at 75% volume to allow addition ofvarious adjuvants. Example 47 homogeneous suspension (4 mg/mL) at 25 and100 μg doses, R848 (1 mg/mL) at 25 μg dose and CpG1826 (10 mg/mL) at 25μg dose were added to the alum formulations or to F-subunit proteinwithin 2 hours prior to immunization. Balb/c mice were immunizedintramuscularly on days 0 and 14 with 100 μL immunization volume (50μL/leg). Mice were bled retro-orbitally on day 28. ELISA titers weremeasured by serial dilutions of the sera on F-subunit protein adsorbedto the substrate. Neutralizing titers against RSV virus were measuredbased on the reduction in plaque numbers when incubated with variousserum dilutions. FIRSV, R848 and CpG were used as the benchmarks in thestudy.

As shown in FIG. 9A, Example 47 suspension increased the total antibodytiters for all samples, and increases the neutralizing titers when theF-antigen was adsorbed to Al—H. FIG. 9A shows both the total anti-Fantibody (ELISA-kappa) and neutralizing antibody (PRNT60) titres.Highest neutralizing titers were observed for the 25 μg dose of Example47. Furthermore, addition of the Example 47 homogeneous suspension alsoenhanced IgG2a isotype switching with RSV F antigen alone or formulatedwith Al—H.

B. Study 2

Similar results were obtained from another study. As shown in FIG. 9B,Example 47 homogeneous suspension increased the total antibody titersfor all samples, and increased the neutralizing titers when theF-antigen was adsorbed to Al—H. Furthermore, addition of the Example 47suspension also enhanced IgG2a isotype switching with RSV F antigenalone or formulated with Al—H.

Example (VII) Investigation of Strain Coverage for MenB Formulations

The in vivo efficacy of Example 47, delivered as homogeneous suspensionat a dose of 25 μg/mouse, was evaluated using a MenB model. The studieswere performed in two mouse strains, one study used CD1 mice and theother used C57/BL6 mice. The evaluation was carried out with a modified5CVMB in which the GNA2091-GNA1870 fusion protein was replaced by the“936-10A-10A” protein disclosed in International Patent Application No.PCT/IB2010/002260 (filed Aug. 27, 2010, claiming priority from U.S. Ser.No. 61/237,576) (SEQ ID NO: 126 therein; SEQ ID NO: 4 herein). Themodified 5CVMB was delivered with Al—H. The MenB antigens were testedfor in vivo immunogenic potency using a SBA assay.

CD1 mice (Experiment I) or CD57/BL6 mice (Experiment II) were immunizedintramuscularly on days 0 and 14 with 100 μL immunization volume (50μL/1 eg) containing 10 μg of the three MenB polypeptides and 25.0 μg ofExample 47 homogenous suspension. Mice were bled retro-orbitally on day28 and SBA titers were measured. Alum/3MenB* with 25% of the standardclinical OMV dose was used as a benchmark. In this example, 3MenB*refers to a MenB vaccine made from the following three separatepolypeptides: GNA2132-GNA1030, 936-10C-10C and NadA.

Table 7 summarizes the immunization of CD1 mice (Experiment I)

TABLE 7 Groups adult mice 6/group 1 Alum 2 Alum/3MenB* 3 Alum/3MenB* +Example 47 (25.0 μg) homogeneous suspension 5 Alum/3MenB* + ¼ D-OMV(1.25 μg) Immunization Protocols: for i.m. immunization: injection of100 μl (2 × 50 μl) per mouse Number of immunizations: 2 Schedule forimmunization: day 0, 14 Bleed Days: −1, 13, 28

Table 8 summarizes the immunization of C57/BL6 mice (Experiment II)

TABLE 8 Groups adult mice 6/group 1 Alum 2 Alum/3MenB* 3 Alum/3MenB* +Example 47 (25.0 μg) homogeneous suspension 5 Alum/3MenB* + ¼ D-OMV(1.25 μg) Immunization Protocols: for i.m. immunization: injection of100 μl (2 × 50 μl) per mouse Number of immunizations: 2 Schedule forimmunization: day 0, 14 Bleed Days: −1, 13, 28

Alum/3MenB* formulations (groups G2, G3, G5 in Table 9) were prepared at75% volume, and Example 47 homogeneous suspension was added in order toreach a 20 μg dose, within 2 hours prior to immunization. The additionof Example 47 homogeneous suspension to Alum/3MenB* did not alter thequality of the formulations in terms of pH and osmolarity as the valueswere consistently within an acceptable range for preclinical andclinical evaluations. The Example 47 homogeneous suspension wascompatible with the antigens used and the endotoxin content was lessthan 5 EU/dose. Table 9 shows that the standard formulations of MenBwere well adsorbed on Alum (>95%) both alone and formulated with OMV.Formulations with Example 47 showed a desorption of the antigens afteraddition of the homogeneous suspension of Example 47, but adsorptionremained >90%.

TABLE 9 Formulation Characterization Overview Ags Osmolarity AdsorptionmOsm/ml pH G1 Alum 1st imm (Exp. I) n/a 245 7.0 2nd imm (Exp. I) n/a 2607.0 1st imm (Exp. II) n/a 282 7.0 2nd imm (Exp. II) n/a 265 7.0 G2Alum/3MenB* 1st imm (Exp. I) >95% 296 7.3 2nd imm (Exp. I) >95% 278 7.41st imm (Exp. II) >95% 293 7.4 2nd imm (Exp. II) >95% 290 7.2Alum/3MenB* G3 Example 47 1st imm (Exp. I) >90% 294 7.3 2nd imm (Exp.I) >90% 280 7.4 1st imm (Exp. II) >90% 279 7.3 2nd imm (Exp. II) >90%285 7.3 Alum/3MenB* + G5 ¼ D-OMV 1st imm (Exp. I) >95% 258 7.2 2nd imm(Exp. I) >95% 270 7.3 1st imm (Exp. II) >95% 285 7.3 2nd imm (Exp.II) >95% 280 7.3

Tables 10-13 show bactericial titers against five different strains ofNeisseria meningitides after immunization with formulations comprisingthe three polypeptides combined with (2) Al—H alone, (3) Al—H+250 mcg/mlExample 47 or (5) Al—H and MenB outer membrane vesicles.

TABLE 10 serum bacterial antibody (SBA) analysis of Experiment I (CD1nice) Exp. I CD1 NZ98/ Group Immunization MC58 254 961-5945 UK355 5-99 1IM <16 <16 <16 <16 <16 2 IM <16 <16 <16 <16 <16 3 IM <16 <16 <16 <16 <165 IM <16 <16 <16 <16 <16

TABLE 11 serum bacterial antibody (SBA) analysis of Experiment II(C57/BL6 mice) Exp. II C57/BL6 NZ98/ Group Immunization MC58 254961-5945 UK355 5-99 1 IM <16 <16 <16 <16 <16 2 IM <16 <16 <16 <16 <16 3IM <16 <16 <16 <16 <16 5 IM <16 <16 <16 <16 <16

TABLE 12 serum bacterial antibody (SBA) analysis of Experiment I (CD1nice) Exp. I CD1; Post 2nd NZ98/ Group Immunization MC58 254 961-5945UK355 5-99 1 Alum/PBS <16 <16 <16 <16 <16 2 Alum/3MenB* 2048 64 1024 <648192 3 Alum/3MenB* + 8192 1024 16384 2048 >32768 Example 47 (250 mcg/ml)5 Alum/3 MenB* + D-OMV 4096 1024 2048 128 >32768

TABLE 13 serum bacterial antibody (SBA) analysis of Experiment II(C57/BL6 mice) Exp. II C57/BL6; Post 2nd NZ98/ Group Immunization MC58254 961-5945 UK355 5-99 1 Alum/PBS <16 <16 <16 <16 <16 2 Alum/3MenB* <64<64 <64 <16 128 3 Alum/3MenB* + 512 128 2048 <16 32768 Example 47 (250mcg/ml) 5 Alum/3 MenB* + D-OMV 128 1024 64 <16 512

SBA titers for Example 47 formulations were higher than for Alum/3MenBbaseline, and were comparable to Alum/3MenB+D-OMV. As shown in Tables10-13, addition of Example 47 homogeneous suspension (Group 3) led to asuperior breadth of vaccine coverage when compared to the Alum/3MenBgroup (Group 2), and better or comparable coverage compared to the groupformulated with OMV (Group 5).

The amino acid sequences of NadA, GNA2132-GNA1030, GNA2091-GNA1870, and936-10A-10A are presented below.

SEQ ID NO: 1 (Amino acid sequence for NadA)ATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADHDTRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVG SEQ ID NO: 2(Amino acid sequence for GNA2132-GNA1030)MASPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGGQDMAAVSEENTGNGGAAATDKPKNEDEGAQNDMPQNAADTDSLTPNHTPASNMPAGNMENQAPDAGESEQPANQPDMANTADGMQGDDPSAGGENAGNTAAQGTNQAENNQTAGSQNPASSTNPSATNSGGDFGRTNVGNSVVIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDGKNDKFVGLVADSVQMKGINQYIIFYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPSKGEMLAGTAVYNGEVLHFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGDGLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGSGGGGATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDIQIEAAKQ SEQ ID NO: 3(Amino acid sequence for GNA2091-GNA1870)MVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYDRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATRARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRGSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ SEQ ID NO: 4(Amino acid sequence for 936-10A-10A)MVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYDRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATRARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRGSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDLGGEHTAFNQLPDGKAEYRGTAFGSDDAGGKLTYTIDFTKKQGNGKIEHLKSPELNVELASAEIKADGKSHAVILGDVRYGSEEKGSYSLGIFGGRAQEVAGSAEVKTVNGIRHIGLAAKQGSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDLGGEHTAFNQLPDGKAEYRGTAFGSDDAGGKLTYTIDFTKKQGNGKIEHLKSPELNVELASAEIKADGKSHAVILGDVRYGSEEKGSYSLGIFGGRAQEVAGSAEVKTVNGIRHIGLAAKQ

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supercede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

The invention claimed is:
 1. A composition comprising an aluminum salt and a homogeneous suspension comprising (a) a Benzonaphthyridine compound of Formula I or Formula II:

pharmaceutically acceptable salt, pharmaceutically acceptable solvate, individual isomer or mixture of isomers thereof; (b) a surfactant, and (c) a suspension agent that is different from the surfactant, wherein the suspension is stable for at least about four weeks at 4° C., wherein the homogenous suspension was produced by high pressure homogenization and wherein: R⁴ is selected from H, halogen, —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂, —NHN(R⁹)₂, —SR⁷, —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷, —LR⁸, —LR¹⁰, —OLR⁸, —OLR¹⁰, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ are each optionally substituted with 1 to 3 substituents independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸, —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂, —S(O)₂R⁸, —S(O)R⁸, —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸; each L is independently selected from a bond, —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene of L are each optionally substituted with 1 to 4 substituents independently selected from halogen, —R⁸, —OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂; R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl groups of R⁷ are each optionally substituted with 1 to 3 R¹³ groups; each R⁸ is independently selected from H, —CH(R¹⁰)₂, C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each optionally substituted with 1 to 3 substituents independently selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹, —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹², —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂; each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸, —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆ heteroalkyl and C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆alkyl that together with N they are attached to form a C₃-C₈heterocycloalkyl, wherein the C₃-C₈heterocycloalkyl ring optionally contains an additional heteroatom selected from N, O and S, and wherein the C₁-C₆ alkyl, C heteroalkyl, C₃-C₆ cycloalkyl, or C₃-C₈heterocycloalkyl groups of R⁹ are each optionally substituted with 1 to 3 substituents independently selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹, —C(O)OR¹¹, —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂; each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are optionally substituted with 1 to 3 substituents selected from halogen, —R⁸, —OR⁸, —LR⁹, —LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸, —CO₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂; R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R¹¹ and R¹² are each optionally substituted with 1 to 3 substituents independently selected from halogen, —CN, R⁸, —OR⁸, —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸, —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸, —S(O)₂N(R⁹)₂, —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and C₁-C₆haloalkoxy; or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken together with the N atom to which they are attached form an optionally substituted C₃-C₈heterocycloalkyl ring optionally containing an additional heteroatom selected from N, O and S; each R¹³ is independently selected from halogen, —CN, —LR⁹, —LOR⁹, —OLR⁹, —LR¹⁰, —LOR¹⁰, —OLR¹⁰, —LR⁸, —LOR⁸, —OLR⁸, —LSR⁸, —LSR¹⁰, —LC(O)R⁸, —OLC(O)R⁸, —LC(O)OR⁸, —LC(O)R¹⁰, —LOC(O)OR⁸, —LC(O)NR⁹R¹¹, —LC(O)NR⁹R⁸, —LN(R⁹)₂, —LNR⁹R⁸, —LNR⁹R¹⁰, —LC(O)N(R⁹)₂, —LS(O)₂R⁸, —LS(O)R⁸, —LC(O)NR⁸OH, —LNR⁹C(O)R⁸, —LNR⁹C(O)OR⁸, —LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂, —LNR⁹S(O)₂R⁸, —LC(O)NR⁹LN(R⁹)₂, —LP(O)(OR⁸)₂, —LOP(O)(OR⁸)₂, —LP(O)(OR¹⁰)₂ and —OLP(O)(OR¹⁰)₂; each R^(A) is independently selected from —R⁸, —R⁷, —OR⁷, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸, —NR⁹C(O)N(R₉)₂, —NR⁹C(S)N(R⁹)₂, —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸, —C(O)CH₂C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸, —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸, —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂, —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸, —CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸; or in Formula I two adjacent R^(A) substituents on the ring to which they are bonded form a 5-6 membered ring that contains up to two heteroatoms as ring members; n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7 or 8; each m is independently selected from 1, 2, 3, 4, 5 and 6, and t is 1, 2, 3, 4, 5, 6, 7 or
 8. 2. The composition of claim 1, wherein the homogeneous suspension comprises about 0.1% to about 10% surfactant.
 3. The composition of claim 1, wherein the homogeneous suspension comprises about 0.1% to about 10% suspension agent.
 4. The composition of claim 1, wherein the homogeneous suspension comprises about 0.5 mg/mL to about 50 mg/mL Benzonaphthyridine compound.
 5. The composition of claim 1, wherein the surfactant is polysorbate
 80. 6. The composition of claim 1, wherein the suspension agent is a viscosity-enhancing suspension agent.
 7. The composition of claim 6, wherein the viscosity-enhancing agent is carboxymethyl cellulose.
 8. The composition of claim 1, wherein at least about 50% of the suspension particles have a diameter of about 10 μm or less.
 9. The composition of claim 1, wherein at least about 50% of the suspension particles have a diameter of about 2 μm or less.
 10. The composition homogeneous of claim 1, wherein the Benzonaphthyridine compound is: 2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenyl)propan-2-ol; 2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine; ethyl 4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylbenzoate; 2-(4-(dimethylamino)phenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine; 2-(4-methoxyphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine; or 2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol, or pharmaceutically acceptable salt, pharmaceutically acceptable solvate, individual isomer or mixture of isomers of any of the foregoing.
 11. A method of producing the composition of claim 1, comprising: (a) mixing a Benzonaphthyridine compound according to claim 1, with a surfactant and a suspension agent; (b) homogenizing the mixture of step (a) under pressure, wherein the pressure is between about 15,000 psi and about 20,000 psi to produce the homogenous suspension; and (c) adding the aluminum salt to the homogenous suspension.
 12. The composition of claim 1, wherein the Benzonaphthyridine compound is 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine or pharmaceutically acceptable salt, pharmaceutically acceptable solvate, individual isomer or mixture of isomers thereof.
 13. The composition of claim 1, wherein the suspension comprises: a) between about 0.1% to about 10% of the surfactant; b) between about 0.1% to about 10% of the suspension agent; and c) between about 0.5 mg/mL to about 50 mg/mL of the Benzonaphthyridine compound, wherein the Benzonaphthyridine compound is in particle form and at least about 50% of the particles have a diameter of about 10 μm or less.
 14. The composition of claim 13, wherein: a) the surfactant is selected from perfluorooctanoate, perfluorooctanesulfonate, sodium dodecyl sulfate, ammonium lauryl sulfate, sodium laureth sulfate, alkyl benzene sulfonate, a fatty acid salt, cetyl trimethylammonium bromide, cetylpyridinium chloride, polyethoxylated tallow amine, benzalkonium chloride, benzethonium chloride, dodecyl betaine, cocamidopropyl betaine, coco ampho glycinate, alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), a copolymer of poly(ethylene oxide) and poly(propylene oxide), octyl glucoside, decyl maltoside, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide DEA, Pluronic F-68, polysorbate 20, polysorbate 80, dodecyl dimethylamine oxide, or a combination thereof; and b) the suspension agent is selected from carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, xanthan gum, sodium alginate, carrageenan, tragacanth, potato starch, guar gum, fumed silica, citronellol, geraniol, dihydro mercinol, linalool, nerol, rhodinal, alphaterpineol, beta-citronellol, rhodinol, citronella nitrile, carvone, fenchone, menthol, isoborneol, or a combination thereof.
 15. The composition of claim 14, wherein the surfactant is polysorbate 80 and the suspension agent is carboxymethyl cellulose.
 16. A composition comprising an aluminum salt and a homogeneous suspension comprising: (a) (i) 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, individual isomers or mixture of isomers thereof; (ii) 2-(4-methoxy-2-methylphenethyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, individual isomers or mixture of isomers thereof; or (iii) 2-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethanol, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, individual isomers or mixture of isomers thereof; and (b) a surfactant, and (c) a suspension agent that is different from the surfactant, wherein the suspension is stable for at least about four weeks at 4° C. wherein the homogenous suspension was produced by high pressure homogenization.
 17. The composition of claim 16, wherein the homogeneous suspension comprises about 0.1% to about 10% surfactant.
 18. The composition of claim 16, wherein the homogeneous suspension comprises about 0.1% to about 10% suspension agent.
 19. The composition of claim 16, wherein the homogeneous suspension comprises about 0.5 mg/mL to about 10 mg/mL 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine.
 20. An immunogenic composition comprising an antigen and the composition of claim
 16. 21. A method of generating an immune response in a subject, comprising administering to said subject the immunogenic composition of claim
 20. 22. A method of producing the composition of claim 16, comprising: (a) mixing 2-(2,4-dimethylphenethyl)benzo[f][1,7]naphthyridin-5-amine with a surfactant and a suspension agent; and (b) homogenizing the mixture of step (a) under a high pressure that is between about 15,000 psi and about 20,000 psi to produce the homogenous suspension; and (c) adding the aluminum salt to the homogenous suspension.
 23. The composition of claim 16, wherein the suspension comprises: a) between about 0.1% to about 10% of the surfactant; b) between about 0.1% to about 10% of the suspension agent; and c) between about 0.5 mg/mL to about 50 mg/mL of the Benzonaphthyridine compound, wherein the Benzonaphthyridine compound is in particle form and at least about 50% of the particles have a diameter of about 10 μm or less.
 24. The composition of claim 23, wherein: a) the surfactant is selected from perfluorooctanoate, perfluorooctanesulfonate, sodium dodecyl sulfate, ammonium lauryl sulfate, sodium laureth sulfate, alkyl benzene sulfonate, a fatty acid salt, cetyl trimethylammonium bromide, cetylpyridinium chloride, polyethoxylated tallow amine, benzalkonium chloride, benzethonium chloride, dodecyl betaine, cocamidopropyl betaine, coco ampho glycinate, alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), a copolymer of poly(ethylene oxide) and poly(propylene oxide), octyl glucoside, decyl maltoside, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide DEA, Pluronic F-68, polysorbate 20, polysorbate 80, dodecyl dimethylamine oxide, or a combination thereof; and b) the suspension agent is selected from carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, xanthan gum, sodium alginate, carrageenan, tragacanth, potato starch, guar gum, fumed silica, citronellol, geraniol, dihydro mercinol, linalool, nerol, rhodinal, alphaterpineol, beta-citronellol, rhodinol, citronella nitrile, carvone, fenchone, menthol, isoborneol, or a combination thereof.
 25. The composition of claim 24, wherein the surfactant is polysorbate 80 and the suspension agent is carboxymethyl cellulose. 